jbei year 9 annual report final · november 1, 2016 contains proprietary information . jbei 2 year...

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

YEAR 9 ANNUAL REPORT

NOVEMBER 1, 2016

CONTAINS PROPRIETARY INFORMATION

J B E I 2 Y e a r 9 A n n u a l R e p o r t

TABLEOFCONTENTS1. Overview.........................................................................................................................................................3

2. FeedstocksDivision....................................................................................................................................42.1. Introduction...............................................................................................................................................................42.2. ScientificProgress...................................................................................................................................................42.3. MajorResearchHighlights....................................................................................................................................82.4. MajorResearch&PersonnelChanges..............................................................................................................82.5. CollaborativeResearch&IndustrialInteractions........................................................................................82.6. ImpactsofResearch................................................................................................................................................92.7. JBEIFacilitiesandProcesses................................................................................................................................92.8. LinkagestoFuturePlans.......................................................................................................................................9

3. DeconstructionDivision.........................................................................................................................103.1. Introduction............................................................................................................................................................103.2. ScientificProgress................................................................................................................................................103.3. MajorResearchHighlights.................................................................................................................................143.4. MajorResearch&PersonnelChanges...........................................................................................................143.5. CollaborativeResearch&IndustrialInteractions.....................................................................................153.6. ImpactsofResearch.............................................................................................................................................153.7. JBEIFacilitiesandProcesses.............................................................................................................................153.8. LinkagestoFuturePlans....................................................................................................................................16

4. FuelsSynthesisDivision.........................................................................................................................164.1. Introduction............................................................................................................................................................164.2. ScientificProgress................................................................................................................................................164.3. MajorResearchHighlights.................................................................................................................................214.4. MajorResearch&PersonnelChanges...........................................................................................................214.5. CollaborativeResearch&IndustrialInteractions.....................................................................................224.6. ImpactsofResearch.............................................................................................................................................224.7. JBEIFacilitiesandProcesses.............................................................................................................................224.8. LinkagestoFuturePlans....................................................................................................................................22

5. TechnologiesDivision.............................................................................................................................235.1. Introduction............................................................................................................................................................235.2. ScientificProgress................................................................................................................................................235.3. MajorResearchHighlights.................................................................................................................................265.4. MajorResearch&PersonnelChanges...........................................................................................................265.5. CollaborativeResearch&IndustrialInteractions.....................................................................................275.6. ImpactsofResearch.............................................................................................................................................275.7. JBEIFacilitiesandProcesses.............................................................................................................................275.8. LinkagestoFuturePlans....................................................................................................................................27

6. Bibliography...............................................................................................................................................28


1. Overview In Year 9, JBEI had 92 publications that are referenced throughout this report, out of a total of 602 publications since the program’s inception. In the past year, JBEI researchers received 20 awards, including the 2015 Samson - Prime Minister’s Prize for Innovation in Alternative Fuels for Transportation (Israel), the 2015 L’Oréal USA for Women in Science Fellow, the Berkeley Lab Director's Award for Early Scientific Career Achievement and election to the Amer-ican Academy of Arts & Sciences. This year JBEI recorded 40 records of invention (ROI) disclosures (252 total), filed 22 patent applications (146 total), had 11 patents issued (20 total), executed 12 licensing agreements (80 total), and initiated 2 funds-in projects (CRADAs) with industry partners. In Year 9, JBEI has been highlighted over 188 times in the press and has hosted more than 104 tours of its facility in Emeryville, CA. JBEI’s integrated research programs are inherently cross-divisional in nature and bring multiple disciplines together to address key problems in JBEI’s feedstocks to fuels efforts, as indicated by the fact that ~45% of the Year 9 publications involved multiple teams at JBEI. JBEI’s research program has played a leading role in developing the scientific underpinnings for the conversion of feedstocks to advanced biofuels and renewable chemicals. Year 9 highlights from the four JBEI Divisions include: Feedstocks Division:

• Discovered UDP-arabinose, UDP-glucuronic acid/galacturonic acid, and UDP-glucosamine transporters and determined their role in cell wall biosynthesis

• Engineered model plants with stacked traits for 50% decrease in lignin, 100% increase in galactan, 20% de-crease in xylan, increased wall density, and improved drought tolerance

• Engineered and characterized sorghum and switchgrass with improved saccharification • Developed two novel approaches to create dominant low lignin and high-saccharification traits • Identified new arabinosyl transferase, galactosyl transferases, acetyltransferases and feruloyltransferases in-

volved in cell wall biosynthesis • Determined drought tolerance of cell wall mutants

Deconstruction Division:

• Developed and demonstrated several variants of a one-pot approach to biomass conversion based on the use of biocompatible and renewable ionic liquids (a.k.a. bionic liquids)

• Demonstrated efficient methods to depolymerize lignin streams generated by the ionic liquid process • Discovered the importance of a GH12 enzyme in the hydrolysis of crystalline cellulose • Revised and released the metagenomics data analysis tool, MaxBin v2.0 • Characterized enzymes in the lignin β-aryl ether cleavage pathway from Sphingobium sp SYK-6 • Developed an engineered strain of Rhodosporidum toruloides that is capable of growth on sugars and lignin-

derived intermediates 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%); engineered E. coli strain with ~5 g/L methyl ketones in fed batch fermentation

• Engineered PKSs for improved expression and function towards the production of new biofuels in a range of organisms.

• Established multi-omics capabilities that facilitated metabolic engineering of biofuel-producing organisms • Discovered tolerance mechanisms to enable improved production of isopentenol and limonene. • Discovered a native E. coli gene that provides the foundation for a IL tolerant strain to express IL tolerant cel-

lulases and converts unsaccharified sugars from IL containing hydrolysate to target fuels. • Developed a Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae

Technologies Division:

• Established high-throughput analysis of plant and microbial metabolites and enzyme activities • Designed and established chips for rapid screening enzyme functions • Established high-throughput proteomics pipelines for biofuels systems biology • Combined cryo-electron tomography and simulations to develop a new model for the plant cell wall. • Use high resolution structural methods to study biofuel pathway enzymes involved in lignin synthesis and

degradation • Further developed the Experiment Data Depot to support proteomics and metabolomics workflows


2. Feedstocks Division

2.1. 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. 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 facili-tate deconstruction for sugar production and to provide a source of lignin for conversion to high-value products. These approaches enable translation of high value traits to bioenergy crops through conventional breeding and ge-netic engineering.

2.2. Scientific Progress 2.2.1. Plant Systems Biology Nucleotide sugar biosynthesis: In order to manipulate polysaccharide biosynthesis, not only must glycosyltransfer-ase (GT) activity be regulated, but so must the proteins responsible for providing substrates for the GT. In collabora-tion with the University of Saitama and the University of Cambridge, we have identified and characterized two pro-teins which boost GDP-Mannose biosynthesis (KONJAC1 and 2). This not only increases substrate availability for glucomannan biosynthesis, but also for vitamin C biosynthesis [1]. In collaboration with the University of Mel-bourne, we have investigated the roles of the different UDP-Xylose synthases (Arabidopsis has 6 isoforms) and shown unexpectedly that it is the cytosolic isoforms (UXS3, 5 and 6), as opposed to the Golgi isoforms (UXS1, 2 and 4), that supply the substrate for xylan biosynthesis in the Golgi [2]. Development of screen for GT modulators: We and others have hypothesized that there are non-catalytic proteins in the Golgi that regulate polysaccharide biosynthesis. We developed a bioinformatics screen to identify non-GT pro-teins, which are co-expressed with mannan synthesis genes. A candidate list of 15 was established (Pick and Morti-mer, unpublished), and all were shown to be Golgi-localized using the rapid transient expression assay system pre-viously developed by the Plant Systems Biology group [3]. The proteins were then screened for interactions with the mannan synthases using (a) a yeast two hybrid screen and (b) an in planta luciferase assay, which was developed specifically for looking at Golgi-localized proteins by the Cell Wall Biosynthesis group [4]. Proteins that were posi-tive in at least one assay were selected for further characterization. Those that were positive in both were prioritized. Two homozygous independent T-DNA lines in each of these candidates have been isolated, and characterized as null mutations, and these plants will be characterized in FY2017. Glycosyltransferases: The engineering of plant polysaccharides requires extensive knowledge of the GTs involved. Whilst the mannan synthases have been identified (the CSLAs), it is not clear whether they require a transporter for the provision of substrate (as has been reported in the literature) or whether, like other GTs such as the cellulose syn-thases, they translocate the substrate across the membrane as part of chain synthesis. Using tagged proteins ex-pressed in Pichia, our protease protection data point to a protein topology with an active site in the cytosol, and therefore there is no requirement for a GDP-Glc or GDP-Man Golgi transporter (Mahboubi and Mortimer, un-published). We have also characterized GMT1, a member of GT64, and shown it is specific for glycosylinositol-phosphorylceramide (GIPC) glycosylation. Plants disrupted in this Golgi-localized protein show normal hemicellu-lose and pectin in their cell walls, but have disrupted cellulose deposition (Fang et al. under revision). We are now working to understand the mechanism underlying this phenomenon. Tools for cell wall analysis: We have continued to develop methods for studying cell wall biosynthesis. In particu-lar, we have focused on methods to improve the sub-fractionation of cellular compartments (including the Golgi) for proteomics studies of plant tissues [5-8]. We have also completed the building of a custom chamber for growing


plants in a 13CO2 environment, which will expand our solid state NMR cell wall analysis capability from one-dimensional (which is poorly resolved and difficult to interpret) to multi-dimensional. In FY2017, we will grow plants for analysis in this system, and work in cooperation with the Deconstruction Division to support understand-ing of the molecular nature of recalcitrance and how our engineering approaches impact cell wall nano-architecture. Translation of knowledge to bioenergy crops: Many proposed bioenergy crops are poorly studied, and in collabora-tion with Washington State University we investigated the effects of light and nitrogen on the photosynthetic effi-ciency of Miscanthus, and how this impacts biomass deposition [9]. We are also building on our proteomics study of switchgrass [10] to work with the JGI to use proteomics for the validation of their switchgrass genome assembly (Mortimer, Schwessinger, Heazlewood, unpublished), and these data are available on jbei.org/research/jbei-data.

2.2.2. Cell Wall Biosynthesis Substrate transport and glycan biosynthesis. The Cell Wall Biosynthesis team is working closely with the Systems Biology team on the characterization of nucleotide sugar transporters. In FY2016, we published our work on the GDP-fucose transporter [11, 12]. The single GFT1 (GONST4) transporter is essential and simultaneously provides GDP-fucose for protein glycosylation, xyloglucan and pectin. This contrasts our previous observations with UDP-Gal/UDP-Rha transporters [13] and UDP-Xyl transporters [14], which show specificity in vivo indicating substrate channeling. In FY2016, we have also submitted a publication on a UDP-GlcA / UDP-GalA transporter and investi-gated several additional transporters. UDP-GlcA transport is a key step in cell wall biosynthesis because the transport of UDP-GlcA is a prerequisite for the synthesis of UDP-GalA and UDP-Ara, as well as for most of the UDP-Xyl. These substrates are required for synthesis of essentially all the non-cellulosic polysaccharides in the wall. UDP-Xyl is synthesized both in the Golgi lumen and in the cytosol, hence UDP-Xyl transport was thought not to be essen-tial. However, knocking out the three cytosolic UDP-Xyl synthases led to strong phenotypic effects and decreased xylan biosynthesis [2]. Clearly, the cytosolic UDP-Xyl synthesis is more important for xylan biosynthesis, in agree-ment with the strong xylan phenotype of UDP-Xyl transporter mutants. Apparently, the Golgi synthesis of UDP-Xyl is more important for biosynthesis of xyloglucan and UDP-Ara. A key objective of JBEI is to develop technologies and resources to rapidly advance our understanding of cell wall biosynthesis. In FY2016 we have identified and characterized several new GTs. A DUF-246 family GT-like gene was identified by coexpression analysis, and we showed that the encoded protein, Pectic ArabinoGalactan synthesis-Related (PAGR), is required for male fertility and the biosynthesis of pectic arabinogalactans [15]. Another GT was determined to be a galactomannan galactosyltransferase with an important function in seeds [16]. The most abun-dant non-cellulosic polysaccharide is xylan, and we have continued our studies to understand the biosynthesis of this complex and important polymer. At least four proteins are required to synthesize the xylan backbone, IRX9, IRX10, IRX14 and IRX15 (or their paralogs IRX9L, IRX10L, IRX14L, and IRX15L) [17]. We have previously shown that expression of a non-functional version of IRX9 or IRX14 did not affect xylan accumulation in the wild-type back-ground [18]. In contrast we have now found that by expressing a non-functional version of IRX10 in the wild type we can suppress xylan biosynthesis. This provides a method for tissue-specific downregulation of xylan in bioenergy crops. The mechanism of the suppression is under investigation, but presumably the dominant negative effect is due to complex formation between IRX9, IRX14 and IRX10. The GUX proteins are xylan glucuronosyltransferases, and we have previously shown that the closest homolog, IPUT1, is a glucosonosyltransferase required for biosynthesis of inositol phosphoryl ceramide sphingolipids, a major class of plasma membrane lipids in plants [19]. Loss-of-function mutants in IPUT1 could not be recovered because the protein is essential for male transmission. To better understand this phenomenon, we complemented the mutant with a pollen-specific promoter driving the IPUT1 gene [20]. In this way, we were able to recover viable plants ho-mozygous for the iput1 mutation. The plants are severely dwarfed and have large changes in sphingolipid composi-tion. We were also able to show that the iput1 pollen germinates normally and pollen tube growth is not affected. However, the iput1 pollen is unable to respond to signals from the ovule, thus pollen tube guidance is severely im-paired. Another gene involved in pollen function is DPW2, which encodes an acyltransferase in rice [21]. The trans-ferase is involved in the biosynthesis of cutin. Our interest in this group of acyltransferases is founded in their in-volvement in lignin biosynthesis and the esterification of grass xylans with ferulic and p-coumaric acid. Characterization of cell walls. We have developed an efficient pipeline for the characterization of cell walls, which we have used in studies of mutants as well as a range of other plants. In FY2016 we published a very thorough study of the composition of cell walls in different tissues and developmental stages in rice [22]. The biochemical changes were correlated with the expression of key glycosyltransferase genes. This has led to the identification of multiple candidate genes for ongoing studies of biosynthesis of grass cell walls. In another study, we investigated the cell wall composition of several different sugarcane varieties, selected based on their differences in lignin content and sacchar-ification [23]. We found that recalcitrance positively correlated with lignin and xylan content, and negatively corre-lated with both mixed-linkage glucan content and highly substituted xylans. In a final study, we used immunofluo-rescence microscopy to profile cell walls in stomata in wild-type plants and pectin methylesterase mutants [24]. The


study showed surprising levels of heterogeneity in the walls of different cell types in the leaf, and demonstrated that deesterification of pectin is required for stomatal function. 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 determine the impact of the cell wall alterations on drought tolerance. We have previously engineered plants with altered cell wall composition that would improve downstream processing by providing lower recalcitrance, decreased acetate or increased C6/C5 sugar ratio. While our plants were shown to have excellent growth properties in non-stressed conditions, it was not investigated if they would show an altered response to stress. Surprisingly, the majority of the plants we have studied, including plants with low xylan and/or low lignin content show improved recovery after drought. The mechanism behind this is still unclear, but we have seen that the plants induce drought-response genes faster than the control plants. Arbuscular mycorrhizae are extremely important for the uptake of nutrients and water, and it is therefore important to ensure that plants with altered cell walls are not impaired in these interactions. We have established a Medicago-Rhizophagus-Rhizobium model system to investigate these interactions, and have identified a number of cell wall proteins and gly-cosyltransfeases that are important for the productive colonization of roots with Rhizophagus. Most of this work is still ongoing, but we have published two papers in FY2016 on the role of arbuscular mycorrhizal fungi in the uptake of nutrients in soils contaminated with heavy metals [25, 26], and one paper on the cross-talk between the symbiotic signaling pathway and abiotic stress tolerance [27].

2.2.3. Grass Genetics Kitaake genome assembly; annotation, sequencing, and analysis of 1,500 rice mutants; KitBase. Rice serves as a model for other grass species, including switchgrass and sorghum. To facilitate the identification of genes regulating bioenergy traits, we generated a mutant population containing over 7,000 M1 lines using fast-neutron (FN) mutagen-esis in Kitaake, an early flowering rice variety, with a rapid generation time of nine weeks. To date, we have se-quenced over 1,500 mutants in collaboration with JGI. Using a newly established sequence analysis pipeline [28], we identified 91,513 mutations, affecting 32,307 genes (58% of all rice genes). Mutation types include single base substi-tutions, deletions, insertions, inversions, translocations, and tandem duplications. We have observed a high propor-tion of loss-of-function mutations in the mutant collection. To make this mutant collection and associated data pub-licly accessible to the research community, we constructed KitBase (http://kitbase.ucdavis.edu/). The whole-genome sequenced mutant population together with KitBase and other open access resources [29] enable rapid func-tional genomic studies in grasses, which differ from the usual approaches by generating mutants with transfor-mations [30]. Switchgrass annotation and phylogenomics analysis. Switchgrass, a fast growing, perennial C4 grass, is considered a prime lignocellulosic feedstock. From our sequenced switchgrass BAC clones and the newly released switchgrass genome, we have identified 1,949 glycosyltransferases, 1,273 glycosyl hydrolases, and 97 lignin biosynthesis-related genes. We have also shown that overexpression of a rice acyltransferase gene named OsAT10 [31] in switchgrass sig-nificantly altered the content of cell wall-bound phenolics, ferulic acid and p-coumaric acid. Saccharification assays show that the engineered switchgrass lines exhibit a 30% increase in the sugar yield. These plants are currently being propagated for field testing in the spring of 2017. Identification and characterization of sorghum TILLING mutants altered in cell wall traits. Sorghum’s small and fully sequenced genome (~730Mb) make it an attractive model for functional genomics studies of C4 grasses. Diverse sorghum inbreeding lines and mutant populations make it an ideal system for the study of the traits that are im-portant in perennial cellulosic biomass crops. Recently, our collaborator has made a whole-genome sequenced EMS-mutagenized sorghum TILLING population. Using this mutant population, we have identified multiple mutants related to cell wall biosynthesis. One of the mutants is mutated in the gene SbCSLF6, which is homologous to the rice CSLF6 gene. In rice, CSLF6 is required for biosynthesis of mixed-linkage glucan (MLG), an important C6 polysaccha-ride in the cell wall. When the rice CSLF6 gene was expressed under the control of a senescence-associated promoter in Arabidopsis thaliana, we obtained up to four times more glucose in the matrix cell wall fraction, and up to a 42% increase in saccharification compared to control lines without any defects in biomass accumulation [32]. To study the effects of CSLF6 in sorghum, we have identified and characterized two sorghum TILLING mutants, cslf6-1 and cslf6-2. The mutants display reduced MLG contents as well as morphological phenotypes similar to those in the rice cslf6 mutants, including reduced height and grain yield. Genetic analysis and saccharification assays on candidate mutants. We screened the whole-genome sequenced fast-neutron (FN) mutant population for alterations in cell wall saccharification and plant immunity. We identified and characterized 15 mutants altered in cell wall composition, immunity, and development. We isolated genes corre-sponding to mutations and showed that the genes encode two cell wall localized cysteine rich receptor kinases [33], a novel regulatory microRNA, a Dicer-like protein, a kinesin-4 protein controlling grass cell wall composition, and a RNA polymerase subunit or antiphosphatase protein. We carried out in-depth analysis of one of the mutants called


rcs481. Mutant rcs481 shows increased homogalacturonan content and reduced plant growth. Our preliminary data suggest that the rcs481 mutant is more susceptible to the bacterial pathogen, Xanthomonas oryzae pv. oryzae [34-36]. The enhanced susceptibility of the rcs481 mutant cosegregates with a 30kb deletion, which contains four genes en-coding cellulose synthase-like family D4 (CslD4) [22], two calmodulin-binding proteins, and a hypothetical protein. Another mutant FN2572 has a 4-bp frameshift mutation in the CslD4 gene and displays similar phenotypes to rcs481. These results suggest that the mutation in the CslD4 gene in mutants rcs481 and FN2572 is responsible for the cell wall- and immunity-related phenotypes.

2.2.4. Cell Wall Engineering Lignin engineering. Based on our previous lignin engineering results [37, 38] our focus remained on developing novel dominant technologies to reduce and manipulate lignin content and composition, respectively, to reduce over-all biomass recalcitrance and facilitate its valorization, without adversely affecting plant biomass yield. We complet-ed our study on the identification and production of a bio-inhibitor of hydroxycinnamoyl transferase (HCT), an im-portant enzyme in the lignin pathway that controls the phenylpropanoid flux toward the production of two major lignin monomers: coniferyl and sinapyl alcohols. In collaboration with the Technology Division, we crystalized HCT protein from switchgrass and demonstrated that protocatechuic acid binds to HCT catalytic site [39]. In addition to a lignin reduction, engineered lines accumulate free protocatechuic acid that could be easily and rapidly converted into muconic acid with microbes. The latter would then be sold as a co-product that would reduce the overall cost of biofuel production. The lignin engineering portfolio was completed with the development of a novel dominant ap-proach that focuses on reducing the availability of AdoMet (S-adenosylmethionine), which is the cofactor used by COMTs (caffeic acid O-methyltransferase) and cell wall methyltransferases in lignifying tissues [40]. More than 10% of the carbon stored in the lignin is derived from AdoMet, which makes lignin sink in the secondary cell wall. There-fore, we expressed an AdoMet hydrolase from Coliphage T3 (AdoMetase) in the secondary cell wall to reduce the availability of AdoMet. Engineered plants exhibit a ~30% lignin reduction which correlates to a saccharification im-provement greater than 25% without a yield penalty, compared to control plants. Since this approach targets OMT enzymes, it makes it compatible with previously developed technologies designed to reduce the production of caffeoyl-CoA such as that of HCT activity inhibition [37, 39]. Therefore, we expect that our latest technology could be used to further reduce lignin content or support lignin composition changes. Finally, in the context of manipulating lignin composition to improve the biofuels production yield, produce advanced biofuels directly in planta, or accu-mulate ionic liquid precursors, we screened a set of BADH acyltransferases that could be potentially used to opti-mize lignin composition using our yeast-based screen approach [39, 41]. With this approach we identified several enzymes that could be used to manipulate lignin composition and enrich it with biofuel molecules such as 2-phenylethanol and isopentanol. These molecules would be stored on the lignin backbone as esters so they can be col-lected in a cost-effective manner [42]. Development of molecular tools to support rapid and precise engineering of energy crops. Stacking of multiple genes or traits through DNA assembly is essential for many synthetic biology tools, metabolic engineering projects, and development of improved energy crops [43]. Even if we are able to efficiently stitch DNA fragments together, there are still many challenges (e.g. size, cost, time, and assembly method flexibility). Therefore, to support the Feed-stocks Division and many other plant scientist groups, we developed a versatile and robust DNA assembly system named jStack, which utilizes yeast homologous recombination as a core technique for DNA assembly into plant transformation vectors [44]. We demonstrated that it can be used to assemble multigene constructs, some larger than 20kb; screen for optimal pathways for the production of biofuels (bisabolene) in planta; isolate and transfer cross-species genomic gene clusters; and stack multiple cell wall traits to improve biomass quality and sugar yield. We also completed the development and characterization of a new two-component repressor system based on endoribonu-cleases and their respective cogni-tion sequences for plant engineer-ing [43, 45]. This device works by inserting a cognition sequence in the 5’UTR of mRNAs in order to render them sensitive to specific endoribonucleases, thus repressing export and translation of mRNAs after cleavage. We demonstrated that this regulatory device was functional in monocot and dicot plant species, supporting an easy translation from model to crop plants; showed that it can be used to repress transgene expression by >400-fold; and used it to synchro-nize transgene repression. In addi-

Figure 1. JBEI QsuB technology for engineering low lignin and improved deconstruction has been transferred to switchgrass, sorghum, and poplar. Several sorghum lines showed up to 50% reduction in lignin and 100% increase in sugar yield after hot water pretreatment.


tion to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Translation of cell wall traits to biofuel crops. We have made significant progress in the translation of cell wall traits developed in model species (rice and Arabidopsis). We independently transferred three cell wall traits into switchgrass: a low lignin content trait (QsuB trait) based on the depletion of HCT substrate (shikimate) and produc-tion of HCT inhibitor (protocatechuate) [37, 39]; low lignin DP based on the production of monolignols forming lig-nin end-chains (HCHL trait [46]); and an increased ferulate/p-coumarate ratio trait (AT10 trait) based on the overex-pression of the acyltransferase AT10 [47]. The saccharification efficiency of biomass derived from switchgrass plants harboring the QsuB or AT10 trait is enhanced. The analysis of the HCHL trait is still in progress. None of the plants showed any growth defect in the greenhouse. Several individual poplar lines harboring the low lignin trait (QsuB trait) were generated in collaboration with Dr. S. Mansfield (U. of Vancouver). More than 10 individual poplar lines were transferred to the greenhouse and will be analyzed in FY17. Finally, in collaboration with Ceres, Inc, we gener-ated several sorghum lines with the low lignin trait (QsuB trait). Some of the lines show up to a 50% reduction in lig-nin, resulting in drastic sugar release improvements (up to 2-fold; Figure 1).

2.3. 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, UDP-GlcA/UDP-GalA, and UDP-Ara transporters

Isolation of transporter – transferase complexes and ki-netic characterization of substrate channeling

Validated two key cell wall traits in switchgrass and one in sorghum

Transfer of best lines to field and technology licensing

Identification of plant cell wall proteins involved in mycor-rhizal interactions in Medicago

Determine mechanism and compare effects in bioenergy crops

Identified genes for regulating polysaccharide substrate availability

Stack these with other traits to regulate biomass composi-tion

Identified and characterized three new glycosyltransferases Determine potential for use in biomass improvement Two novel dominant approaches to manipulate lignin con-tent or composition without impacting plant biomass yield

Implementation of lignin valorization traits while reduc-ing biomass recalcitrance

Identified and characterized wall-based drought tolerance genes in Arabidopsis

Determine biological mechanism and assess potential in bioenergy crops

Identified cslf6 sorghum mutants Assess growth and saccharification phenotypes of the two mutants

Identified 15 mutants altered in cell wall composition and immunity

Isolate and characterize genes corresponding to the mu-tations

Sequenced and analyzed 1500 rice mutants Sequence 700 more mutants to achieve 70% saturation, Complete Kitaake genome assembly

Developed a robust technology for rapid DNA assembly and transfer of traits in plants

Implementation of this technology to engineer energy crops with multiple energy traits

2.4. Major Research & Personnel Changes The research goals and directions of the Feedstocks Division in Year 9 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 remain important focus areas. Other than the expected regular turnover of post-doctoral fellows, no major personnel changes have taken place in FY2016.

2.5. 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. Barry

Switchgrass, Arabidopsis, and rice sequencing

GLBRC J. Ralph Lignin modification HudsonAlpha Institute of Biotechnology

J. Grimwood, J. Schmutz Switchgrass genomics

Univ of Copenhagen, Den-mark

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, A. Bacic Nucleotide sugar transporters and linkage analysis Technical Univ. of Denmark 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

mutants Univ. of British Columbia S. D. Mansfield Translation of lignin competitive pathway approach Forest Genetics International 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 University of Liege A. Richel Lignin valorization USDA sorghum Tilling Team, Texas

Z. Xin

Translation of traits to sorghum

2.6. Impacts of Research Our cell wall engineering efforts have been highly successful, and additional publications and patents have been made in Year 9. Afingen Inc. is a JBEI startup located in Emeryville and working on commercialization of the artifi-cial positive feedback loop technology to develop improved switchgrass varieties and yeast strains. Afingen has four full time employees and is funded through two SBIR grants. The switchgrass plants are in field 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 work-ing 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.

2.7. JBEI Facilities and Processes The Feedstocks Division has all of the equipment and personnel required to conduct the work. The JBEI facilities, procurement processes, and Work Planning & Control processes are all performing optimally.

2.8. Linkages to Future Plans In Year 10, we will continue the stacking of genes for improved biomass composition and saccharification, as well as immunity and drought tolerance. We have observed synergy between transferases and proteins involved in sub-strate biosynthesis and transport, and our gene stacks require the introduction of up to ten genes. After testing in model plants, we will transfer the most promising stacking approaches to sorghum and switchgrass in Year 10. In Year 9 we have developed switchgrass with improved saccharification and we will evaluate these plants in the field in Year 10. At the same time we will produce sufficient biomass to test the entire feedstocks-to-fuels pipeline at JBEI at a relatively large scale. We will continue to develop functional genomics, bioinformatics and synthetic biology tools. In particular, we will continue sequencing rice mutants to achieve a high level of saturation in the mutant pop-ulation and develop tools to optimize transgene expression and trait robustness. These resources establish a firm foundation for reverse and forward genetics analyses to identify genes controlling cell wall properties, stress toler-ance and carbon allocation. We have built a system for 13C-labeling of plant material and we will use this to investi-gate metabolic fluxes, identify bottlenecks in allocation of carbon to the cell wall, and support cell wall structural characterization and recalcitrance with the cooperation of the Technology Division. We will continue to work with the Deconstruction and Fuel Synthesis Divisions to evaluate the impact of the engineered plants on fermentable sug-ar production and biofuel yields, and to manipulate lignin such that it can be better valorized. In addition to evaluat-ing downstream conversion, we will investigate the tolerance of engineered plants to drought and pathogens.

J B E I 1 0 Y e a r 9 A n n u a l R e p o r t

3. Deconstruction Division

3.1. 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.

3.2. Scientific Progress 3.2.1. Biomass Pretreatment Process integration for biomass to biofuels using biocompatible ionic liquids (ILs). The integration of IL pretreat-ment with enzymatic saccharification and microbial fermentation is challenging due to the toxicity of the ILs current-ly used for pretreatment, requiring extensive water wash. In FY16, we published several variants of the a one-pot, integrated process for the production of fuels directly from lignocellulose without removal of IL or any other additional separation or post-treatment operations prior to saccharification and fermenta-tion. In one instance, to over-come the pH mismatch and cytotoxicity issues with com-mercially available enzyme mixtures and wild type fer-mentation hosts, we screened ILs for biocompatibility and employed CO22 to implement an integrated one-pot pre-treatment, saccharification and fermentation process us-ing cholinium lysine ([Ch][Lys]). As shown in Fig-ure 2, high ethanol yields (85%) were achieved using wild type yeast (S. cerevisiae) in the presence of [Ch][Lys] [48]. This approach resolves several of the most significant obstacles towards the realiza-tion 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 provided by the Fuels Synthesis Division 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.

Figure 2. Schematic of (left) the one-pot conversion of switchgrass using [Ch][Lys] and (right) the ethanol yields obtained as a function of IL recycle and reuse [48].


We also 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 re-sult, a relatively high titer of ethanol (over 40 g/L) was produced with a one-pot approach for the first time. This in-tegrated 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 reduce annual operation cost by 40%, compared to the conventional JBEI IL process [49]. 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 in lignin analytics required for this project. 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 [Ch][Lys]) using 1D and 2D NMR techniques. Our results show multiple H-bonding interactions between the dilignol and IL. For both [C2C1Im][OAc] and [Ch][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 [Ch][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 [Ch][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 [Ch][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. Depolymerization of aqueous IL-processed lignin stream. Switchgrass was pretreated with [Ch][Lys], followed by enzymatic saccharification using the one-pot approach. The lignin-enriched residue stream was recovered after sac-charification and subjected to catalytic transfer hydrogenoloysis using isopropanol as a hydrogen-donor solvent at 300 °C . From the industrial standpoint, catalytic transfer hydrogenation is an attractive alternative for high-pressure catalytic hydrogenation with molecular hydrogen. During transfer hydrogenoloysis of lignin, hydrogen-donating solvents readily generate or transfer hydrogen molecules in situ, which can subsequently promote hydrogenation reaction. When depolymerized with ruthenium catalyst for 3 hrs, it resulted in high liquid yield, up to 65.5 wt% with a significant amount of monomers (27 % in liquid product) and low char formation. The compositional analysis shows that alkyl-substituted phenols, including 4-ethylphenol, 4-ethyl-2-methoxyphenol and 2,6-dimethoxy-4-ethylphenol are the main products (Figure 2). Overall, hydrogenolysis of biorefinery lignin presents a promising ap-proach for a conversion into valuable products, which can be used directly or as platform chemicals for further up-grading.

3.2.2. Enzyme Optimization Structure—function of lignin demethylases (LigM) involved in bacterial metabolism of lignin fragments. Aryl demethylation is essential for metabolism of aromatics in lignin utilizing bacteria such as Sphingobium ssp but, due to the energetic stability of aromatic rings, is particularly challenging. In Sphingobium SYK6, LigM-catalyzed demethyla-tion is essential for utilization of vanillate and 3-O-methylgallate. LigM-catalyzed demethylation enables further modification and ring opening of aromatic compounds, which is essential for conversion and utilization of common aromatic industrial waste products such as lignin derived from biomass pretreatment processes. Our results provide the first structure – function and mechanistic details of how LigM catalyzes demethylation of vanillate and in the process transfers the methyl group to tetrahydrofolate. We characterized the kinetics and mechanism of LigM and determined it functions optimally at temperatures between 20 and 40 °C and pH between 6 and 8 and uses a bi-bi sequential mechanism in which vanillate binds first followed by binding of tetrahydrofolate and subsequent transfer of the methyl group. LigM has no significant homology to any protein with a structure in the PDB. We solved the crystal structure of the apo form of LigM by X-ray crystallography and used molecular docking, quantum chemistry


calculations, and Atoms in Molecules methods, to localize both vanillate and tetrahydrofolate in the LigM active site and define the interactions among substrates and amino acid residues in LigM that stabilize substrate binding. This study advances our understanding of demethylation and its reaction requirements, defining LigM as a new branch in the demethylase family tree. Database of lignin depolymerizing and modifying enzymes (LigDB). Knowledge of their substrates and products, conditions for optimum function, kinetics and synergies with other lignolytic enzymes is critical to understanding and controlling lignin depolymerization and conversion to either desired end products or intermediates suitable for uptake by host organisms engineered to convert them into valuable products. While some of this knowledge is scat-tered throughout the literature and in databases such as the CAZy, which now contains the FOLy database of fungal oxidative enzymes, the peroxibase database of peroxidases and the LCCED database of laccases, there is currently no comprehensive database of well-characterized fungal and bacterial lignolytic enzymes. We have begun the process of designing and populating a database of enzymes that catalyze lignin depolymerization, lignin modification, lignin transport and lignin metabolism. Our current database is designed to provide the following information: enzyme name(s), enzyme classification or family, reaction type, substrates, products, cofactors, reaction scheme, activators, inhibitors, Km, turnover number (kcat), Ki, pH optimum, melting temperature, temperature optimum, assay condi-tions, NCBI accession number, source organism, protein sequence, cross-reference accession number (BRENDA, ExPASy, etc.), structural information (PDB ID), recombinant production strategy, purification methods, long-term storage and stability and selected references. Our database, while under design and prototyping, is in the form of a spreadsheet and contains enzymes from both fungal and bacterial sources involved in lignin depolymerization and metabolism. Our plans for building out LigDB are to 1) develop web scraping tools for mining the literature for pre-viously characterized enzymes, 2) to screen assay additional lignolytic enzymes using both model substrates and our previously developed lignin film assay, 3) to work with the Technology Division to turn the spreadsheet version of our database into a web-based database, and 4) when completed, to share the database with the outside community through publications and by posting on JBEI.org. We currently have an archive of approximately 150 genes coding for putative lignolytic enzymes and synthesized for us by the JGI. The biggest hurdle to assaying these enzymes has been expressing them in soluble form, and we are working with the Fungal Biotechnology group to resolve these issues. Multicomponent enzyme mixtures. Our previous multi-component mixture of a heterologously expressed defined set of enzymes included three enzymes to catalyze conversion of insoluble cellulose from ionic liquid pretreated bi-omass to glucose. This set of enzymes included Cel5A (endoglucanase from T. maritima), CSac (cellobiohydrolase from C. saccharolyticus), and A5IL97 (beta-glucosidase from T. petrophila). This minimal enzyme mixture produced approximately 30% glucose yields from [C2C2Im][OAc] pretreated switchgrass. To increase glucose yields, and to achieve the release of xylose as well, we screened an additional 153 enzymes, spanning GH families 1, 3, 5, 6, 9, 10, 12, 55 as well as AA9. Enzymes were expressed in E.coli, and the resulting purified proteins were tested for activity using a panel of soluble substrates across a range of pH values and temperatures to determine their optimal activity profiles. Approximately one third of these enzymes had detectable levels of soluble expression, and unfortunately, none of the lytic polysaccharide mono-oxygenases (LPMO, AA9 family) enzymes expressed in soluble form in E. coli and we have given several of these enzymes to the Fungal Biotechnology group to test for expression in A. niger. Six-teen enzymes were selected based on their activity levels and temperature and pH profiles for inclusion in mixture experiments. Using these enzymes, we have developed a four-component enzyme mixture that produces 35% glu-cose yields and 35% xylose yields at 70 °C from [C2C1Im][OAc] pretreated. The final mixture consists of a GH10 fami-ly endo-xylanase from Thermotoga maritima, a GH9 family endoglucanase from T. maritima, a CBM3-GH5 family con-struct from Caldicellulosiruptor saccharolyticus and a GH1 family b-glucosidase from Thermotoga petrophila. Enzyme – ionic liquid interactions. The activity of the family 5 glycoside hydrolase from Thermotoga maritima (Tm_Cel5A) decrease linearly with increasing concentration of the ionic liquid [C2C1Im][OAc], reaching zero activity at 50% (~1.2 M) IL concentration. In control experiments, the kinetics of Tm_Cel5A in aqueous solutions of sodium acetate at 70 °C with the substrate 4-nitrophenyl-β-D-cellobioside (pNPC) showed an approximate 20 unit decrease in Vmax in going from 125 mM to 1000 mM sodium acetate with no change in Km. In solutions of [C2C1Im][OAc], Vmax dropped rapidly (~20 units) when moving from 15mM to only 125 mM [C2C1Im][OAc], while Km increased by 45 units over the same [C2C1Im][OAc] concentration range. Taken together, this data suggested inhibition by [C2C1Im], and molecular docking experiments show [C2C1Im] can occupy the Tm_Cel5A active site by ring stacking with a tryp-tophan residue associated with cellulose binding. We are currently performing combined quantum mechanical and molecular mechanical studies to investigate the interactions between the IL and the enzyme that alter Km and Vmax of Tm_Cel5A. In the absence of any IL, quantum chemistry geometry optimization located the stationary points on the gas phase potential energy surface corresponding to reaction, transition and product states. The resulting contin-uum solvation corrected surfaces showed that the approximate catalytic effect of the enzyme (∆∆g‡) is 9.1 kcal mol-1, achieved by stabilization of the transition states. In the next phase we will investigate the effects of the IL on ∆∆g‡.

3.2.3. Microbial Communities


Mechanisms of microbial ionic liquid tolerance. During this year, we demonstrated that communities tolerant to ionic liquids tetrabutylphosphonium chloride and tributylethylphosphonium diethylphosphate could be cultivated when grown on switchgrass [53]. We also crystallized an ionic liquid-responsive regulator, EilR, from ‘Enterobacter lignolyticus and structurally characterized of [C2mim][OAc] to the protein, providing a mechanism for regulator, which was previously demonstrated to control expression of an MFS-1 pump that conferred ionic liquid tolerance to E. coli. In collaboration with UC-San Diego and Technical University of Denmark (DTU) we performed adaptive evo-lution on E. coli in the presence of ionic liquids [C2mim][OAc] and [C4mim]Cl. Strains of E. coli were adapted to grow on up to 10% [C2C12Im][OAc] and the mutations causing the tolerance phenotype are being analyzed by resequencing of the adapted strains. Cellulase discovery from microbial communities. Cultivation of cellulolytic consortia revealed that communities dominated by Thermobispora bispora, a thermophilic actinomycete, had high levels of hydrolytic activity on crystalline cellulose. Comparative proteomics in collaboration with EMSL revealed that the level of GH12 in the supernatant was a critical factor in the crystalline cellulose activity an unexpected result as GH12 is an endoglucanase and the levels of the canonical exoglucanases, GH48 and GH6, as well as the AA10 protein did not correlate with increased activity on crystalline cellulose. Heterologous expression of these proteins demonstrated that the GH12 had the highest specific activity for cellulose hydrolysis of the three glycoside hydrolases and NIMS-based enzymatic assays revealed that the GH12 hydrolyzed cellulose by a random mechanism. This mechanism diverges from the classical mechanisms of cellulase action on cellulose and suggests that there may be additional hydrolytic mechanisms, espe-cially in bacterial systems [54]. Previous annual reports have described the identification of the enzymatic compo-nents of Jtherm, an IL/thermotolerant protein mixture isolated from a microbial community, which were organized into protein complexes that were distinct from cellulosome isolated from anaerobic Clostridia. In this year, we recov-ered the full length sequences from the active proteins in the complexes and demonstrated that they were in a gene cluster. Unexpectedly, the proteins contained catalytic domains and multiple CBM domains, resembling glycoside hydrolases expressed by hyperthermophilic anaerobes in the Calidicellulosiruptor genus. The cellulases responsible for the Jtherm activity were heterologously expressed in E. coli and the activity of the individual proteins on cellulose characterized. Lignin deconstruction and aromatic metabolism. We have developed a method 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 [55]. The size fractionated, soluble lignin fractions were used to isolate two Penicillium species that could grow on lignin fractions ranging from 1-100 kDa. The proteins involved in the depolymerization of these lignin fractions are currently being investigated. Combined experimental and metabolic modeling studies on aro-matic metabolism in Ralstonia eutropha and Pseudomonas putida demonstrated that the metabolism of lignin-derived aromatics (p-hydroxybenzoate, p-coumarate) generates overflow metabolites at high concentrations (>5 g/L), sug-gesting that adaptive evolution and metabolic engineering strategies will need to be employed to improve the carbon conversion efficiency of aromatic metabolism. Despite this inefficiency, strains of Ralstonia eutropha and Pseudomonas putida were engineered to produce medium chain methyl ketones from both sugars and aromatics. Bioinformatics. MaxBin 2.0, an automated binning tool for recovery of genomes from multiple metagenomic sam-ples was released and published in FY16 [56]. MaxBin 2.0 was entered into the CAMI challenge (http://www.cami-challenge.org/) to compare to other automated binning algorithms. MaxBin has been downloaded >2000 times and referred in >50 publications since it was made available in 2014. The automated algorithm was used to reconstruct the genome of a deeply branching, uncultivated member of the Chlorobi, OPB-56, found in compost enrichment cul-tures growing on ionic-liquid pretreated switchgrass and closely related genomes were recovered from multiple, publically available metagenomic data sets obtained from thermal springs [57]. Genomes (>400) were also recovered from solid state enrichments growing on progressively higher levels of ionic liquid and high salt environments (San Francisco Bay salterns), which may harbor ionic liquid tolerant microbes and enzymes.

3.2.4. Fungal Biotechnology Improving heterologous production of cellulases and ligninases in A. niger. Previous Aspergillus niger mutagenesis and screening efforts resulted in the isolation of many mutant strains that over-produced a heterologously expressed bacterial β–glucosidase, cellobiohydrolase or endoglucanase target from the high performance JTherm cocktail (Gladden et al.). The genomes of these strains were resequenced in collaboration with JGI and a number of high pri-ority gene candidates were identified from bioinformatics analyses, mostly with point mutations. One of these genes, a sugar transporter known as mstC was deleted in an A. niger strain expressing the J03 β-glucosidase, and this ΔmstC strain resulted in 2.5x the production of J03 of the parent strain as measured by β-glucosidase activity. Successful heterologous expression of more than 30 bacterial and basidiomycete cellulases in A. niger with enzyme properties similar or identical to those expressed in bacteria, without hyperglycosylation issues, has demonstrated the utility of the A. niger heterologous enzyme expression system. We have issued an additional challenge to the system this year


by transforming A. niger with a dozen codon optimized ligninase genes identified by the Enzyme Optimization team as highly desirable enzymes for study individually and as components of cocktails. PCR has confirmed the incorpo-ration of most of these targets into the genome of A. niger. The background ligninase activity (laccase, it has no pe-roxidases) in A. niger is low under our culture conditions, so as we move forward with screening for activity it should be clear when we have obtained the correct conditions for expression of ligninases in these strains. In Year 10 we will continue our focus on improving the overall performance of the A. niger platform by deleting amino acid sinks and over-expressing other genes that may be advantageous for higher heterologous protein expression, espe-cially ligninases. Fungal platform for the production of biofuel intermediates from sugars and lignin. Technoeconomic analyses have indicated that deriving value from all of the carbon in biomass, including lignin, will be critical for economical-ly and environmentally sustainable lignocellulosic biorefineries. Fungi are nature’s principal lignin degraders, so it was not surprising but gratifying that we were able to isolate Basidiomycete yeast strains that grew well on lignin monomers. The carotenogenic (tetraterpene synthesizing) and oleaginous yeast Rhodosporidium toruloides emerged this year as our winning platform for terpene production. In collaboration with the Fuels Synthesis Division and Jeff Skerker of UC-Berkeley, we genetically engineered Rhodosporidium toruloides to produce bisabolene by incorporating the bisabolene synthase gene. We have shown that it utilizes glucose, xylose, p-coumaric acid and lignin-rich hydrol-ysates as carbon sources, and produces at least 50-100 mg/L of bisabolene under all of those conditions. Experiments in bioreactors at the ABPDU resulted in titers up to 300 mg/L under further optimized conditions. In Year 10 we will continue this exciting cross-division and extramural collaboration and bolster it with an FY17 JGI-EMSL FICUS pro-ject to use multi-omics approaches aimed at increasing terpene synthesis and understanding potentially novel lignin catabolic pathways and optimized fluxes to intermediates for biofuels and biochemicals.



Developed a one-pot approach to biomass conversion using biocompatible ionic liquids

Determine the impact of engineered crops from Feed-stocks and biofuel hosts from Fuels Synthesis on process efficiency and yield

Demonstrated that active component of Jtherm is a non-cellulosomal complex with unprecedented struc-ture

Reconstitute the complex by heterologous expression of its constituent proteins

Revised and released MaxBin 2.0 Determine how to expand user base of MaxBin 2.0, includ-ing working with DOE KBase

Separated ionic-liquid pretreated lignin into soluble, size-defined fractions; developed and demonstrated approaches for depolymerization of these lignin streams

Demonstrate microbial and enzymatic deconstruction on these defined depolymerized fractions of lignin to increase yield

Demonstrated 50-100 mg/L production of bisabolene on a wide variety of biomass substrates including hexoses, pentoses and lignin monomers using oleagi-nous yeast

Further increasing titer rate and yield to efficiently utilize the carbon from valuable plant biomass substrates in a collaboration with Fuels Synthesis Division

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

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

3.4. Major Research & Personnel Changes The research goals and directions of the Deconstruction Division in Year 9 have largely followed the plan with more focus on the development of biocompatible ionic liquids and demonstrating an integrated biomass conversion pro-cess. Other than the expected regular turnover of post-doctoral fellows, no major personnel changes have taken place in FY2016.


3.5. Collaborative Research & Industrial Interactions


BESC Charlie Wyman, Mark Davis, Mike Crowley, Frederick M. Hahn

Comparison of dilute acid and IL pretreatment; high-throughput screening of biomass; lignin analysis; plant cell wall modeling; glycome profiling of plant cell walls before and after pretreatment

GLBRC Tim Donohue, Bruce Dale, Ven-katesh Balan, Trey Sato, John Ralph

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

Compact Mem-brane Systems

Stuart Nemser Dewatering and recycle of ILs

JGI Kerrie Barry, Sam Deutsch Gene synthesis, metagenomics, expression and character-ization of GH1 enzymes

Environmental Molecular Sci-ences Laborato-ry

Robby Robinson, Ljiljana Pasa-Tolic, Scott Baker, John Cort, Nancy Isern

Metaproteomics, proteomics, metatranscriptomics, HSQC NMR

University of Queensland (AU)

Robert Henry Analysis and pretreatment of eucalyptus species for bio-fuel production

Imperial College (UK)

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

University of Milan

Fabrizio Adani Ionic liquid pretreatment of Arundo donax

Idaho National Laboratory

Vicki Thompson, David Thompson, Chenlin Li

Mixed and densified feedstocks, enzyme mixture opti-mization

ABPDU Todd Pray, Deepti Tanjore, Ning Sun

Scale-up of IL pretreatment and recombinant protein expression

Total Florence Mingardon Conversion of lignin into renewable aromatics UC Berkeley Jeffrey Skerker Engineering R. toruloides for conversion of lignin mono-

mers into biofuels precursors Proionic Roland Kalb Dewatering and recycle of ILs

3.6. 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 10 and beyond. There is one start-up company, Illium Tech-nologies, based on the R&D conducted by the Division, and there are two industry funded CRADAs with POET and Total. MaxBin has been downloaded more than 2,000 times and is being adopted by users in the external scientific community. The discovery of a bacterial non-cellulosomal complex that is very effective at hydrolyzing polysaccha-rides is novel and provides new insights into the role and mechanisms by which bacteria degrade cellulose in the environment.

3.7. JBEI Facilities and Processes The Deconstruction Division has all of the equipment and personnel required to conduct the work. The JBEI facili-ties, procurement processes, and Work Planning & Control processes are all performing optimally. The purchase of a new size exclusion chromatography instrument dedicated to the analysis of lignin is a key addition to JBEI’s analyti-cal infrastructure.


3.8. 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 10 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 in Apsergillus niger. 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 Divisions in the development of enzymes and engineered microbial hosts that can tolerate ILs and syn-thesize valuable biofuels and renewable chemicals from sugars and lignin, and will aggressively pursue the results obtained in red yeast.

4. Fuels Synthesis Division

4.1. Introduction The primary goal of JBEI’s Fuels Synthesis Division is to identify the challenges and develop approaches to enable engineering of microorganisms to efficiently convert sugars to advanced biofuels with properties similar to petrole-um-based fuels. To that end, we are developing fuel synthesis pathways based on the fatty acid, isoprenoid, polyke-tide, and aromatic amino acid biosynthetic pathways. These pathways are being engineered into one or both of two main 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. We also need improved tools to swiftly engineer strains to test production and other desira-ble features in a production strain.

4.2. Scientific Progress 4.2.1. 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, d-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, methyl and ethyl ketones

We have prepared sufficient quantities of several of these fuel targets for fuel property tests and confirmed that they have fuel properties comparable to commercial gasoline or diesel fuels. In FY15, we published a comprehensive re-view of fatty acid-derived and isoprenoid biofuels and bio-based chemicals [58]. 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. 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 ketones, fatty acid ethyl esters, fatty alcohols and fatty alkenes. To further im-prove fatty acid production, we are reprogramming genes in central metabolism to alter the levels of acetyl-CoA, NADH and NADPH. Diesel-range methyl ketones. In 2014, we had made pathway and host engineering modifications to E. coli strains bearing a novel pathway for biosynthesis of C11 to C17 methyl ketones [59]. These modifications resulted in a 160-fold increase in titer in minimal medium (1% glucose) relative to the best strain reported in 2012, 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 fermentation. In FY16, we carried out fed-batch fermentation studies with our best strain from 2014 (EGS1895) and an improved strain. In fed-batch mode, we improved titer to 4.8 g/L, which is by far the highest titer for methyl ketones (other than acetone) yet reported in engineered or native bacteria. In addition, in collaboration with Deconstruction, we investigated methyl ketone production in strain EGS1895 in the presence of ionic liquid (IL) hydrolysate (one-pot process). After testing several ILs, including cholinium α-ketoglutarate ([Ch]2[α-kg]), cholinium lysine ([Ch][Lys]), cholinium phosphate ([Ch]3[PO4]), and ethanolamine acetate ([Ea][OAc]), we found that [Ch]3[PO4] and [Ch]2[α-kg] were the least toxic to E. coli. Strain EGS1895 was capable of tolerating up to 5% w/w of these ionic liquids without significant impact on growth. However, E. coli consumes α-ketoglutarate from [Ch]2[α-kg] as a carbon and electron source. Therefore, we focused our studies of methyl ketone optimization on [Ch]3[PO4] biomass hydrolysates. Exten-sive tests with [Ch]3[PO4]-treated Arabidopsis biomass demonstrated that neither growth nor yield were markedly af-fected in the presence of the IL-treated hydrolysate relative to minimal medium (M9-MOPS) that had comparable sugar composition as the hydrolysate. Ladderanes. Anammox (anaerobic ammonia-oxidizing) bacteria make unusual lipids containing 3 or 5 fused cyclo-butane 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 biosynthesis are un-known. In FY16, we published our extensive in vivo and in vitro studies with candidate ladderane biosynthesis genes (synthesized by JGI) [60]. These genes included putative phytoene desaturase and radical SAM enzymes hypothe-sized to catalyze critical steps in ladderane biosynthesis. Isoprenoid-derived Fuels. Short, branched alcohols and long, branched and cyclic hydrocarbons can be derived from isoprenoids. We engineered the isoprenoid precursor pathways in S. cerevisiae and E. coli. We also engineered these two hosts to produce the following isoprenoid-based fuels: α-Bisabolene. Bisabolane (reduced α-bisabolene; cetane number of 52.6) is a promising diesel and bio-jet fuel candi-date [61]. To improve previously achieved bisabolene titers for in E. coli and yeast strains (~3.5 g/L and ~6 g/L, re-spectively) we have balanced pathway protein level using PCA analysis of proteomics data and production result, and we achieved 1.2 g/L of bisabolene titer in batch fermentation which is approaching 50% of theoretical yield [62]. Limonene, α-pinene, 1,8-cienole, and linalool. We engineered E. coli to produce various monoterpenes as potential jet and diesel fuel precursors by expressing genes encoding codon-optimized geranyl diphosphate (GPP) synthase and vari-ous plant or bacteria derived terpene synthases. Various metabolic engineering efforts have been pursued to im-prove titers and yields, and for example, we improved limonene titers from less than 50 mg/L to 600 mg/L by bal-ancing the pathway from the analysis of proteomics data [62] and addressed other terpenes in prior years. We ex-panded target monoterpenes portfolio and developed engineered strains for 1,8-cineole (titer of 783 mg/L) and linal-ool (titer of 770 mg/L) by introducing chromosomal mutation to improve precursor pool. Isopentenol. Previously, we engineered E. coli to produce the C5 alcohols (isopentanol, 3-methyl-3-butenol, and 3-methyl-2-butenol) [63]. 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) [64]. The study of the pathway bottle-necks using proteomics and metabolomics data further improved the production titer of isopentenol to 2.2 g/L (about 70% of pathway-dependent theoretical yield and 56% of theoretical max) [65]. New IPP-bypass pathways for isopentenol production were proposed and constructed for more efficient isopentenol production, especially in a less aerated condition [66]. High-throughput enzyme screening method has been developed to further improve promis-cuous activity of the key enzyme in the new pathway. With this growth-linked screening, we have improved the effi-ciency of the new pathway over 30%. Polyketide-derived fuels. Polyketides are some of the most diverse molecules known. The enzymes that make them, polyketide synthases (PKSs), perform Claisen condensation reactions between a loaded acyl-ACP intermediate and an a-substituted (H, CH3, C2H5, etc.) malonyl extender unit analogous to fatty acid biosynthesis. By successfully rearranging existing polyketide modules and domains, one could exquisitely control chemical structure from DNA sequence alone. We have begun work on engineering PKSs for production of fuels and have demonstrated produc-tion MKs [67], EKs [67], and alcohols (unpublished) by constructing hybrid PKSs from parts of various PKS that have the targeted chemistries. The lipomycin PKS (LipPKS) is useful for producing short, highly-branched molecules; the borrelidin PKS (BorA4,5,6) is useful for producing longer molecules that would be useful as diesel or jet fuel. De-pending on the final (termination module) on the PKS, it will produce acids, alcohols, alkenes, or ketones. The MKs that we have produced are in the range of molecules useful as gasoline replacements [68].


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, and generated a list of top gene candidates for the toluene synthase. In FY16 we published this work [69] and car-ried out in vitro assays with purified, re-combinant candidate toluene synthase pro-teins. We successfully discovered and confirmed the toluene synthase (which we call GRE, or glycyl radical enzyme) and its cognate activating enzyme (a radical SAM enzyme that we call AE) (Figure 3). This discovery has fundamental relevance in that it is the only known toluene synthase and is only the seventh known glycyl radi-

cal enzyme. It has practical bioenergy significance in that it should enable first-time biochemical synthesis of an aro-matic hydrocarbon fuel for aviation.

4.2.2. 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. In prior year, analyzed the isopentenol production strains based on the correlation of protein levels in each pathway with key me-tabolite levels was used to improve the product yield from 6% to 46% of apparent theoretical yield [64]. We built on this initial result to develop 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% [62]. 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 developed a work-flow to analyze these multi-omics data to more accurately understand and predict the pathway behavior in biofuel producing hosts [70].To understand the effect of the accumulation of IPP, a key intermediate of all isoprenoids biosynthesis, we have designed 3 strains system that showed different IPP-accumulation profiles, collected multi-Omics data including proteomics, metabolomics, and RNAseq at multiple time points, and analyzed the data on E. coli genome scale mod-els to elucidate how the host responds to the stress occurring during high titer biofuel production. 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 have achieved sufficient flux through the DXP pathway to support growth in the absence of added ergosterol. This manuscript is now completed and in peer review.

4.2.3. Synthetic Biology VectorEditor and DeviceEditor software. Both of these software tools have been rewritten from Adobe FLEX (Flash) into javascript, which is extremely timely given the anticipated dropping of support for all Flash applications by all major web browsers by early 2017. While in general little if any new functionality has been developed, these rewrites ensure the continued operation of this JBEI mission-critical software infrastructure going into 2017, and better enable our ability to further develop VectorEditor and DeviceEditor in response to emerging JBEI needs. ICE (Inventory for Composable Elements) repository software. DNA sequence auto-annotation features have been developed and deployed, which enable users to annotate portions of their sequence of interest with corresponding features found within other existing (and identically sub-sequence matching) DNA sequences in ICE. This is very

Figure 3. Phenylacetic acid decarboxylase reaction (left) showing radical intermediate and 13C labeling of phenylacetic acid and tolu-ene, and (right) showing 13C-labeled toluene being produced in an anaerobic assay containing GRE, reconstituted AE, and SAM, but not in a negative-control assay lacking SAM.


important and useful, as legacy JBEI or externally generated DNA sequences provided to current researches may be vastly under-annotated and thus difficult to understand and work with.

4.2.4. Host Engineering Optimization of Carbon source utilization from IL hydrolysates. In a collaboration with the Deconstruction Divi-sion, we discovered and characterized a mutation in the E. coli rcdA gene that endows high tolerance to imidazolium-based ILs. We used a strain with the rcdA mutation to incorporate a plasmid encoding genes for the production of limonene, and showed improved production of limonene in media containing ILs relative to the WT production strain. We also engineered IL tolerant cellulases into this rcdA mutant strain and demonstrated the production of limonene using deconstructed cellulose (Figure 4). Finally, we used IL pretreated switchgrass for the culti-vation and production of limonene using the IL tolerant E. coli strain. In E. coli the use of the rcdA mutant allows production of the bio-jetfuel precursor from IL pretreated Biomass under IL exposure conditions. The WT strains are unable to grow or produce fuels unders these conditions. This study also led to identification of YbjJ, a major facilitator superfamily pump, that is responsible for the primalry imidazolium tolerance phenotypes. We filed a record of invention for the YbjJ gene. The complete study was also published [71]. Optimal Carbon use in S. cerevisiae: In FY16, we completed and published our study that enhanced the uptake and uti-lization of xylose in S. cerevisiae [72]. In FY16, for S. cerevisiae we also addressed the requirement to optimize growth using a true biomass hydrolysate produced by the Biomass Pretreatment Team as the carbon source. We conducted a series of growth assays with range of media containing various batches of cholinium-based IL pre-treated switchgrass. Specifically, we tested yeast strains under many different IL and media conditions, we demonstrated that moving from imidazolium-based to cholinium-based ILs has been the single best improvement for both cultiva-tion and production. We further identified several parameters that impact growth of yeast in choline-based ILs. Pre-liminary tests for Fatty alcohol production were successful. The complete study is being finalized for submission for peer reviewed publication. 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, in FY15 we initi-ated 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. In FY16 we have completed a genome wide survey of native genes, the deletion which provide these two features. To meet this mile-stone we developed a new method to isolate and analyze different subsets of cell populations based on relative levels of membrane protein expression. We call this method FlowSeq, and it combines powerful tools such as fluorescence activated cell sorting (FACS) and next generation sequencing (NGS) to comprehensively discover genetic interrup-tions that change membrane protein expression. Our method uses the barcoded transposon library in E. coli BW25113 (obtained from the Adam Deutschbauer Lab at LBNL) in conjunction with 6 membrane protein fusions, a non-functional membrane protein control, and a soluble protein control. By analyzing the BarSeq data, we have identified several gene candidates spanning multiple functional groups in E. coli that, when deleted, lead to higher levels of the desired membrane protein of interest. These genes are a comprehensive, genome-wide interrogation of gene knock-outs that improve expression of membrane proteins. Expanding strain engineering tools in S. cerevisiae. To address the need that all pathways may eventually require integration into the genome, an integrated project across several JBEI divisions was initiated in FY15 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. In FY16 we completed this the development of this toolkit and the manuscript was sub-mitted for publication. The toolkit includes 23 Cas9-gRNA plasmids, 37 promoters of various strengths and temporal expression profiles, and 10 protein-localization, degradation, and solubility tags. We facilitated the use of these parts via a web-based tool, which automates the generation of DNA fragments for integration. We demonstrated the ap-plicability of this toolkit by optimizing the expression of a challenging but industrially important sesquiterpene syn-

Figure 4: Schematic depiction of the E. coli rcdA deletion chassis that can integrate the expression of IL tolerance cellu-lase and biojet fuel production pathways.


thase. This approach enabled us to diagnose an issue with the synthase solubility, the resolution of which yielded a 25-fold improvement in production. Engineering yeast for production of polyketides. Although S. cerevisiae is widely used for producing fatty acid and isoprenoid-derived fuels and chemicals, its use for production of polyketide-based fuels has been limited because 1) few Type I PKSs have been expressed in S. cerevisiae and 2) S. cerevisiae produces few of the polyketide precursors. Over the last year, we have engineered S. cerevisiae with the pathways for production of propionyl-CoA, methylmalonyl-CoA, butyryl-CoA, isobutyryl-CoA, and hexanoyl-CoA. In addition, we have expressed genes encod-ing the DEBS module 6 and DEBS thioesterase in S. cerevisiae and demonstrated that it is five times more active than the same gene expressed in E. coli. This host should be very useful for producing a variety of polyketide-based fuels. Engineering Streptomyces venezuelae as a host for production of fuels and chemicals. Due to the rich history of Streptomyces as producers of important natural products, this genus of bacteria has recently garnered attention for its potential applications in the broader context of synthetic biology. However, the dearth of genetic tools available to control and monitor protein production precludes rapid and predictable metabolic engineering that is possible in hosts such as E. coli or S. cerevisiae. In an effort to improve genetic tools for Streptomyces venezuelae, we developed a suite of standardized, orthogonal integration vectors and an improved method to monitor protein production in this host. These tools were applied to characterize heterologous promoters and various attB chromosomal integration sites. A final study leveraged the characterized toolset to demonstrate their utility to produce the biofuel precursor bisabolene using a chromosomally integrated expression system. These tools advance S. venezuelae to be a practical host for future metabolic engineering efforts [73].

4.2.5. Quantitative Metabolic Modeling Host engineering through flux analysis. Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. We have used a computational technique developed at JBEI, which com-bines 13C labeling experiments with genome-scale models (2S 13C MFA, [74]), to characterize fluxes, direct metabolic

engineering efforts and increase fatty acid production in S. cerevisiae [75]. The flux profile obtained from this technique showed that any extra acetyl-CoA gained through the addition of an ATP citrate lyase (ACL) was diverted into the malate synthase (MALS) reac-tion (Figure 5) instead of into fatty acid synthesis. Downregulation of MALS resulted in an increase in production of 26%. Knocking out glycerol-3-phosphate dehydrogenase (GPD1) as also suggested by flux analysis resulted in an extra increase in pro-duction of 33%. The final strain produced 780 mg/L of fatty acid, representing an increase of 70% over the initial titer. The same flux modeling technique was used to comprehensibly study an intriguing phe-nomenon in S. cerevisiae: how the presence of galac-tose in a strain with galactose metabolism disabled produces a growth increase which is enhanced by knocking out the Snf1 kinase, in apparent violation of glucose repression conditions. This study was com-pleted and submitted for publication. Finally, 2S 13C MFA is being used in conjunction with OPTFORCE [76] to increase methyl ketone production in E. coli. The initial results show a 3-fold increase by knocking out the acetate kinase (ACK) and the phosphotrans-acetylase (PTA). Pathway engineering through Machine Learning. While previous efforts using simple data mining techniques have been successful [62], improving lim-onene and bisabolene yields by 40% and 200% re-

spectively, we have developed Machine Learning methods to leverage the large amounts of -omics data JBEI can generate. As a proof of principle, we have generated proteomics and production data for 27 strains using an auto-mated fermentation platform and fed these data into four different algorithms: random forest, linear, gaussian, and support vector. The resulting strains improved limonene production by 17% in the first round.

Figure 5. Sankey diagrams obtained from 2S-13C MFA showing all the reactions producing and consuming ace-tyl-CoA, along with their fluxes. The addition of ACL increases the amount of acetyl-CoA being produced but this extra flux, instead of significantly increasing fatty acid production (ACCOACr), is diverted into the malate synthesis (MALS).


Software development. We have renamed our multiomics data visualization tool into Arrowland, and have made it public (https://public-arrowland.jbei.org/). Furthermore, we have collected the methods we have developed and tested for metabolic modeling in JBEI into an open source library that includes several examples from our published work. This tool is now described in a study submitted for publication. Finally, we have developed new data stream input workflows for our fermentation platform (BioLector by m2p-labs) data and proteomics and metabolomics.



Produced FAEE, alkenes, methyl ketones, isopenta-nol, limonene, α-pinene, 1,8-cineole, linalool, α-bisabolene.

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

Multi-omics facilitated metabolic engineering in iso-prenoid pathway.

Expand approach for pathways beyond isoprenoids

Novel IPP-bypass pathway for isopentenol produc-tion under less aerobic condition and development of high throughput screening for improved mutant en-zymes

Demonstration of the efficiency of new pathway under oxy-gen limited condition using fermenter. Further optimization of the pathway with improved PMD enzyme

Achieved 4.8 g MK/L titer in improved methyl ke-tone-producing, E. coli strain and demonstrated in one-pot IL hydrolysates that cholinium phosphate hydrolysate does not negatively affect growth or MK yield.

Further improve performance and scale up production.

Discovered toluene synthase that is a novel glycyl radical enzyme and the first toluene synthase de-scribed.

Characterize novel enzyme and construct E. coli that can synthesize toluene from cellulosic sugars.

Engineered PKSs to produce a variety of short-chain methyl and ethyl ketones

Improved expression to achieve high production

Replaced outmoded FLEX tools including VectorEdi-tor and DeviceEditor with modern javascript coun-terparts. Implemented ICE auto-annotation of DNA sequences.

Support for protein sequence entries in ICE. Integration of DIVA with JGI bioCAD/CAM tools including BOOST.

Engineered S. cerevisiae for production of polyketide precursors; demonstrated production of PKS with high activity

Further improvements in flux needed; complete integration of precursor pathways and engineered PKS needed

Developed expression vectors for Streptomyces venezuelae and demonstrated their use for improving production of bisabolene

Demonstrate use of expression vectors for a variety of other fuels and chemicals.

Development of an E. coli strain for one-pot biofuel production from ionic liquid pretreated cellulose and switchgrass.

Improve titer and yield from such integrated strains.

Developed a CRISPR Cas9 based toolkit for native loci, promoter for use S. cerevisiae to aid with development of optimized production for final tar-gets

Utilize the toolkit to enhance carbon utilization and metabo-lite production pathways.

Utilize the bar-coded, E. coli gene deletion library to comprehensively identify genes that allow improved expression of a membrane protein of interest

Validate the utility of these genes in developing a host that enables great function from desired membrane protein.

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.

Made our multiomics visualization tool, Arrowland, public

Further develop visualization of multiomics data set by making it more intuitive.

4.4. Major Research & Personnel Changes Jay Keasling stepped down as the VP of Fuels Synthesis and Aindrila Mukhopadhyay was appointed the VP of Fuels Synthesis effective Dec 2015, after an extensive search and interview process. The Fuels Synthesis Division also add-


ed a new department, Synthetic Metabolic Pathways, led by Jay Keasling. This new department will focus on engi-neering novel biosynthetic pathways (such as those using polyketide synthases) for the production of drop-in fuels and chemicals. Taek Soon Lee was appointed the DVP for Fuel Synthesis. Beyond the normal and expected turnover of post-doctoral researchers, the only significant personnel change was the hire of a research scientist, Ee-Been Goh as an LBNL Career Research Scientist, and in Synthetic Biology Informatics, software developer Oge Nnadi was re-placed by Elena Aravina.



JGI Deutsch, Simirenko, Oberortner, Tringe

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

JGI Wendy Schackwitz E. coli re-sequencing for spontaneous mutants Penn State Universi-ty

Costas Maranas Full E. coli metabolism and atom transition model

Sandia CRF John Dec Identification and testing of key fuel targets ALS, LBNL Craig Taatjes Combustion chemistry of isopentanol and bisabolane 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, Arbor-gen, NREL

Peter, Rothman, Davis, Sykes

ARPA-E PETRO: Engineering of terpene biofuels in pine

LBNL Adam Deutschbauer Provided E. coli Bar-coded library for membrane protein expres-sion project.

ABPDU Todd Pray, Chenlin Li, Screening of biofuel-producing hosts using IL hydrolysates gen-erated by the ABPDU at the 10L scale.

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

4.6. Impacts of Research Two startups have been founded based on technology developed in the Fuels Synthesis Division. TeselaGen Bio-technology, 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, has 10 full time employees in Emeryville, CA and is funded through several DOE SBIRs. In Year 9, a third startup was founded based on Fuels Synthesis Division technology; Evodia will develop engineered microbes to convert in-expensive, renewable sugars into high-value specialty chemicals. 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 representa-tive fuels from cellulosic sugars were improved dramatically and tested in fed-batch mode and in the presence of IL hydrolysates. Mixed carbon using S. cerevisiae strains were shared using MTAs with industry labs and were inde-pendently tested by them, as well as at the ABPDU. When these strains and fuels are commercialized, they will re-duce the amount of greenhouse gas added to the atmosphere substantially, without needing a change in engines. The Fuels Synthesis Division also developed synthetic biology software tools, most recently including DIVA, which has been adopted by the National University of Singapore.

4.7. JBEI Facilities and Processes The Fuels Synthesis Division has all of the equipment and personnel required to conduct the work. The JBEI facili-ties, procurement processes, and Work Planning & Control processes are all performing optimally. The purchase of the BioLector in FY16 has been a key addition to the JBEI infrastructure.

4.8. Linkages to Future Plans In Year 10 we will optimize biofuel pathways for isopentenol and methyl ketones for production at pilot scale. We will establish the baseline data needed to develop polyketide synthases (PKSs) for methyl and ethyl ketone produc-tion, as well as phenylacetate decarboxylase for toluene biosynthesis. We will use ‘omics tools and correlation analy-sis 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 metabolic 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 Arrowland tool, in collaboration with the Technology Division, to make 2S-13C MFA a routinely used method. We will further develop more sophisticated ma-chine learning tools than PCA for the high-throughput omics data. We will develop support for protein entries in ICE, as well as integrate DIVA with JGI bioCAD/CAM tools including BOOST. We will develop hosts that are more tolerant to biomass inhibitors and fuels, and will screen hydrolysates generated by the Deconstruction Division from engineered and wild type plants produced by the Feedstocks Division, and that are able to metabolize the variety of sugars that result from biomass deconstruction while minimizing byproduct generation. We will investigate the tol-erance and transport systems found to improve isopentenol production and limonene tolerance, in greater detail. We will further 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.

5. Technologies Division

5.1. 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; automating, parallelizing, and miniaturizing procedures that are currently throughput-limiting; and developing new analytical technologies for enzyme activities, biomass, protein, metabolite and cell characterization. The field of biofuels research presents some unique challenges, combining anal-ysis 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 protocols. High-throughput analytical tools are required to test large-parameter spaces for biomass pretreatment and subse-quent enzymatic liberation of sugars including characterization of heterogeneous biomass substrates. Finally, new tools for design, build and test are required to speed up the process of microbial engineering.

5.2. Scientific Progress 5.2.1. Microfluidics We continued to advance the droplet-based microfluidic platform for two applications: 1) metabolic engineering in microbes to optimize production of biofuels, and 2) selection of organisms for IL-tolerance. The key accomplishment this year was integration of all critical steps of transformation and culture in one chip including plasmid addition, heat-shock transformation, addition of selection medium, culture, and phenotypic readout. The chip was validated by transforming a variety of plasmids into E. coli including plasmids containing genes encoding fluorescent proteins GFP, BFP and RFP; plasmids with selectable markers for ampicillin or kanamycin resistance; and a Golden Gate-assembled plasmid. We also demonstrate the applicability of this platform for transformation in other widely used eukaryotic organisms including S. cerevisiae and A. niger. In collaboration with Fuels Synthesis Division, we are im-plementing a CRISPR/Cas9-based method in the chip for combinatorial optimization of chromosomal integration locus and promoter selection, protein localization, and solubility in yeast cells. Towards the eventual scale-up and manufacturing of the microfluidic platform, we also established two strategic partnerships with leading companies. We have partnered with Autodesk, Inc. to integrate design software for metabolic engineering with the microfluidic

chip control software. This will allow a user to seamlessly go from design of pathways to engi-neering them in bacterial and other organisms. We have also partnered with Intel, Inc. to ex-plore fabrication and packaging methods that can be scaled up for large-scale production of our chips. This will be crucial for successful commercialization and dissemination of the chip platform.

5.2.2. Array Based Assays Cost effective deconstruction of biomass into substrates for biofuel production requires high-performance low-cost enzyme cocktails that are active under demanding process conditions.

Figure 6. Lanthanide-chelator complexes enable mass spec-trometry readable droplet barcoding.


Developing these cocktails requires high throughput low-sample volume enzyme activity assays. Earlier this fiscal year, we reported our high throughput platform technologies based on oxime-NIMS technology, automatic biomass handling, robotic liquid handling, substrates spanning cell wall glycosidic linkages, and nanostructure-initiator mass spectrometry (NIMS) [77]. Now this platform of technologies is being routinely used and resulted in two additional publications: In the first, as part of an ongoing collaboration with GLBRC, the oxime-NIMS technology was used for quantitative catalysis studies for CBM3a, CBM6, CBM30, and CBM44 fusion enzymes [77]. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ion-ic-liquid pretreated switchgrass. In the second, as part of a project lead by the JBEI Deconstruction Division, Oxime-NIMS provided rapid analysis of the products of glycosides hydrolases GH12, GH6-exo and GH48 proteins with Av-icel, filter paper, and PASC and enabled determination of the initial rates and products of hydrolysis[54]. This demonstrated that the hydrolysis of the cellulose substrates by GH12 protein is operated by a random mechanism and GH12 was not a processive enzyme. The amount of material required for our current platform which is based on multiwall-plates, makes large-scale in vitro expression cost preclusive. To address this challenge, we have integrated microfluidic and NIMS technologies resulting in a device enabling enzyme kinetic analysis from 150 nanoliter droplets. An invention disclosure has been submitted and a manuscript describing this device and application for GH kinetics is now in review. To enable inte-grated microfluidics-NIMS based screening of combinatorial enzyme screening we designed and demonstrated a lanthanide-chelator based combinatorial barcoding system using a custom chelator with a high lanthanide-affinity enabling barcoding with 39 non-redundant lanthanide isotope between the atomic masses of 136 to 176 (Figure 6). Unique lanthanide barcodes enable tracking each precursor component, such as enzymes, substrates and cofactors, in the generation of combinatorial conditions to determine its relative abundance in the final combined mixture. We have demonstrated our lanthanide-chelator combinatorial barcoding strategy performed a combinatorial cellotetraose digestion assay with enzymes CelEcc_CBM3a (CelE) and β-glucosidase (β-glu) over nine discrete conditions and submitted an invention disclo-sure on this technology.

5.2.3. Proteomic Analysis Rapid proteomic hypothesis testing is crucial to success in many medical and biotechnology research studies. Characterizing and quantifying proteins in microbes contributes to successful meta-bolic engineering for renewable production of biofuels. Much of proteomic research follows two steps: a discovery step to identi-fy proteins of interest followed by subsequent quantitative ex-periments on a targeted subset of proteins, yet the process to bridge these two steps is poorly defined, time-consuming, and difficult. For hypotheses concerning new hosts, new pathway proteins, and strain optimization efforts that involve large quan-tities of samples, high-throughput targeted proteomic are neces-sary. Yet, development of SRM assays for new target proteins is often time-consuming. To overcome this bottleneck, we devel-oped a SRM assay workflow by using data derived from discov-ery proteomics and the retention time calibration methods (Fig-ure 7). This workflow simplifies validation of peptides identified from discovery proteomic experiments and significantly reduces development time of high-throughput quantitative SRM assays (Figure 7a). The workflow is instrument agnostic and performs well by using online resources from other labs to inform SRM target selection. We applied this method to microbes E. coli K-12 BW25113, E. coli DH 5α, Bacillus subtilis 168, Pseudomonas putida KT2440, Coryne-bacterium glutamicum, Agrobacterium tumefaciens EHA1, and Rhodosporidium toruloides that were cultured in house and used to construct a proteome spectral library for each organism. Sub-sequently, we used our E. coli K-12 BW25113 proteome spectral library as an example to assess the workflow by creating sched-uled SRM methods that either target hundreds of proteins in a 20-minute chromatographic gradient or small set of proteins in a

Figure 7. New SRM workflows for high-throughput quantitative assays.


very short LC gradient (2 minutes; Figure 7b, c). We applied this method to characterize 73 proteins from E. coli cen-tral metabolism in over 500 KEIO collection knockout strains grown in two media conditions. Over 3000 samples were analyzed with the two-minute UHPLC-SRM method and over 250,000 individual SRM transition measure-ments were collected and clustered together. These experiments provide a rich dataset for systems biology analysis of E. coli metabolism under different conditions. Working with the Quantitative Metabolic Modeling group in the Fuels Synthesis division and KBase researchers we are analyzing the data to apply it to new metabolic engineering efforts. With the Fuels Synthesis Division, we applied proteomic methods to strains of E. coli that were engineered to identi-fy ladderane biosynthesis proteins [60] and to characterize a key enzyme in toluene biosynthesis [69]. We also helped develop a workflow that integrates metabolomics, proteomics, and genome-scale models of Escherichia coli metabo-lism to study the effects of introducing a heterologous pathway into a microbial host [78]. We also applied targeted proteomics to a chimeric PKS engineered toenable production one of the most widely used commoditychemicals, adipic acid [79]. Targeted proteomics of Acyl-ACP intermediates revealed a bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain.

5.2.4. 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 [80]. We have 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 have used serial sec-tion transmission electron microscopy of cryogenically preserved samples in 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 com-puter-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 applied computational approaches for the simulation of the mechanical properties of cell walls and their macromolecular components, which has resulted in the surprising discovery that plant cell walls are not optimized for mechanical load bearing but for maximal mechanical flexibility and ductility. This approach can also be applied to genetically altered feedstocks in order to 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 modalities that 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 ap-proaches with novel large volume 3D electron microscopy imaging approaches, and found hard X-ray microscopy of heavy-metal stained and resin-embedded samples 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 chal-lenge to image macromolecular resolution details in the respective cell and tissue context. This will ultimately re-quire the development of a map-based database ideally at the model space that can be queried for quantitative vol-umetric information

5.2.5. Structural Biology We continue to pursue crystallographic studies of en-zymes across all divisions within JBEI. Lignin is a com-binatorial polymer comprised of monoaromatic units that are linked together via strong chemical bonds. Alt-hough lignin is a potential source of valuable aromatic chemicals, its recalcitrance to depolymerization presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts. Based on our collaboration with the JBEI Deconstruction Division and researchers at GLBRC we published structural and biochemical studies of the multi-enzyme pathway capable of b-aryl ether bond cleavage by microbes [81, 82]. JBEI researchers in the Feedstocks Division are interested in identifying and characterizing the enzymes responsible for the biosyn-thesis of lignin. They have exploited the substrate flexi-bility of hydroxycinnamoyl-CoA:shikimate hy-droxycinnamoyl transferase (HCT) to reduce lignin con-tent and improve biomass saccharification. We used structural methods to determine the binding of a non-canonical substrate, protocatechuate, in a similar man-ner as shikimate in the active site of the enzyme [83].

Figure 8. Cartoon representation of the effector binding pocket showing the residues involved in van der Waals contact and electrostatic interactions between the EilR repressor and the Malachite Green inducer.


Researchers in the JBEI Deconstruction Division have modified a bacterial repressor protein, EilR, and its target op-erator DNA, eilO, to develop a system for predictable and economical control of gene expression. They have demon-strated that hydrophobic cationic dyes at nM levels efficiently induce transcription in Escherichia coli and distantly related bacteria, Pseudomonas putida, Sinorhizobium meliloti, and Caulobacter crescentus. We have applied structural bi-ology methods to provide atomic level insights into the interactions of the repressor with its operator DNA and with two inducers, malachite green (Figure 8) and crystal violet. Finally, we have collaborated with researchers in the JBEI Fuels Synthesis Division to begin determining the mechanisms and specificity of polyketide synthases (PKS) and fatty acid synthases (FAS), which are capable of producing complex, high-value fuels and chemicals. Initial cryo-EM studies have shown that it is possible to obtain stable FAS complexes for imaging, and also for crystal growth. In all of these studies, 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 PHENIX software developed by researchers at LBNL and elsewhere.

5.2.6. Synthetic Biology Informatics EDD (Experiment Data Depot) repository software. The EDD has been further developed to support proteomics and metabolomics workflows. This is important as it enables the JBEI proteomics and metabolomics teams to deliver data to researchers in a standard and well-structured manner (including linkage to strain information in ICE) that not only preserves the data that will constitute JBEI’s legacy and that enables downstream modeling/analysis tools, but also increases data delivery throughput and accuracy over ad-hoc spreadsheets delivered via email. AgPredictor and CASdesigner grassroots software tools. These two software tools, originally developed by gradu-ate students and postdocs in the Feedstocks and Fuel Synthesis Divisions, respectively, have been re-developed by software developers employing software engineering best-practices, and have been made accessible to the JBEI community (and broader communities post publication) via web-based graphical user interfaces.



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 mi-crofluidic 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 evolu-tion and metabolic path assembly.

Demonstrated the integration of microfluidics with NIMS analysis. Apply automated NIMS characteriza-tion of enzyme activity against 12-substrate panel to support BRC cocktail development efforts. Demon-strate a mass spectrometry readable enzyme bar-coding approach.

Development of high throughput bioanalytical tools to support the analysis of lignin active enzymes.

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

Simulation of mechanical cell wall properties and expan-sion of this approach to feedstock mutants.

Structural and biochemical characterization of the bacterial repressor, EilR, and several different effector molecules, to help develop a highly controlled and versatile system for economical gene expression.

Structural characterization of polyketide synthases and fat-ty acid synthases to engineer their specificity and optimize their activity.

Developed workflows to rapidly target proteins from new engineering hosts and optimize high-throughput data acquisition methods.

Implement workflows to characterize new hosts of interest to JBEI and expand workflows to include metabolomic studies.

Further developed the Experiment Data Depot to support proteomics and metabolomics workflows. AgPredictor and CASdesigner grassroots software tools redeveloped into professional web applications.

Development of an automated software integration testing environment, and redevelopment of an additional grass-roots software tool into a professional web application.

5.4. Major Research & Personnel Changes In Proteomics, Jonathon Vu was hired to implement proteomic sample preparation methods. In Structural Biology Thomas Ellinghaus, a Research Assistant, left to pursue graduate studies in Germany, Ditte Welner a postdoctoral


associate left to pursue research in Denmark, and Giovani Tomaleri joined the group as a Research Assistant. In Syn-thetic Biology Informatics, software developer Jason Eads was replaced by Teresa Lopez.



GLBRC Brian Fox Enzyme libraries from JGI gene synthesis and GLBRC in vitro expression. We are also collaborating on characterization of lignin degrading enzymes using biochemical and spectroscop-ic techniques.

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

Agilent Miniaturized screening platforms for molecular biology

Michigan State University-LBNL

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

5.6. 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. The unique combination of sensitivity, specificity and high-throughput of the NIMS approach was essential in characterizing a library of GH1 enzymes [84]. Both the microfluidic assays and NIMS technology have been critical in collaborations with GLBRC, to characterize lignocellulolytic enzymes [85] [86]. 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 experiments in collaborations. Short chromatography methods for targeted proteomic assays have enabled large-scale studies in-volving many strains, conditions, and replicates across all divisions and with collaborators. The Technology Division also continued to develop synthetic biology software tools, most recently including EDD, and the grassroots tools AgPredictor and CASdesigner.

5.7. JBEI Facilities and Processes The Technologies Division has all of the equipment and personnel required to conduct the work. The JBEI facilities, procurement processes, and Work Planning & Control processes are all performing optimally. In particular, the shared purchase of the cryo-EM detector will solidify JBEI’s access to state-of-the-art analytical instrumentation.

5.8. Linkages to Future Plans Microfluidic technology development will focus on further developing the droplet microfluidic chips to perform highly-parallel DNA assembly for optimization of metabolic pathways to produce fuels and other chemicals. We will also continue to integrate microfluidic chips to NIMS chips towards creating a completely automated pipeline for building and screening metabolic pathways. In addition, this approach can be used to increase the number of condi-tions 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 Func-tional Genomics group will continue to collaborate with all JBEI Divisions to generate methods for high-throughput multi-omic research projects. Efforts will focus on developing high-throughput targeted proteomic workflows for biofuel and PKS pathway optimization. In Physical Analysis we will continue to image plant biomass, microbes for fuel synthesis and microbial communities deconstructing biomass. We will extend our models for the 3D organiza-tion of plant cell walls to the level of whole plants. In Structural Biology, we will continue to collaborate with all JBEI Divisions to generate high-resolution structural information for fundamental understanding of protein function and subsequent engineering. A high priority project is the use of cryo-electron microscopy and crystallography to study the structures of large molecular complexes involved in fatty acid synthesis. In Synthetic Biology Informatics, we will develop an automated software integration testing environment, as well as productionize an additional grassroots JBEI software tool into a web application.


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