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Publications

When it comes to interdisciplinary collaboration, the titles of GLBRC publications speak for themselves. Each new year of operation has seen more publications from multiple labs that span the four Research Areas, accelerating the Center's production of the basic research that generates technology to convert cellulosic biomass to advanced biofuels.

Publications

How willing are landowners to supply land for bioenergy crops in the Northern Great Lakes region?

Scott M. Swinton; Sophia Tanner; Bradford L. Barham; Daniel F. Mooney; Theodoros Skevas

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2017

Land to produce biomass is essential if the United States is to expand bioenergy supply. Use of agriculturally marginal land avoids the food vs fuel problems of food price rises and carbon debt that are associated with crop and forest land. Recent remote sensing studies have identified large areas of U.S. marginal land deemed suitable for bioenergy crops. Yet the sustainability benefits of growing bioenergy crops on marginal land only pertain if land is economically available. Scant attention has been paid to the willingness of landowners to supply land for bioenergy crops. Focusing on the northern tier of the Great Lakes, where grassland transitions to forest and land prices are low, this contingent valuation study reports on the willingness of a representative sample of 1107 private, non-corporate landowners to rent land for three bioenergy crops: corn, switchgrass, and poplar. Of the 11% of land that was agriculturally marginal, they were willing to make available no more than 21% for any bioenergy crop (switchgrass preferred on marginal land) at double the prevailing land rental rate in the region. At the same generous rental rate, of the 28% that is cropland they would rent up to 23% for bioenergy crops (corn preferred), while of the 55% that is forest land, they would rent up to 15% for bioenergy crops (poplar preferred). Regression results identified deterrents to land rental for bioenergy purposes included appreciation of environmental amenities and concern about rental disamenities. In sum, like landowners in the southern Great Lakes region, landowners in the Northern Tier are reluctant to supply marginal land for bioenergy crops. If rental markets existed, they would rent more crop and forest land for bioenergy crops than they would marginal land, which would generate carbon debt and opportunity costs in wood product and food markets. This article is protected by copyright. All rights reserved.

Hybridization and adaptive evolution of diverse Saccharomyces species for cellulosic biofuel production

David Peris; Ryan V. Moriarty; Wiliam G. Alexander; EmilyClare Baker; Kayla Sylvester; Maria Sardi; Quinn K. Langdon; Diego Libkind; Qi-Ming Wang; Feng-Yan Bai; Jean-Baptiste Leducq; Guillaume Charron; Christian R. Landry; Jose P. Sampaio; Paula Goncalves; Katie E. Hyma; Justin C. Fay; Trey K. Sato; Chris T. Hittinger

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2017

Background: Lignocellulosic biomass is a common resource across the globe, and its fermentation offers a promising option for generating renewable liquid transportation fuels. The deconstruction of lignocellulosic biomass releases sugars that can be fermented by microbes, but these processes also produce fermentation inhibitors, such as aromatic acids and aldehydes. Several research projects have investigated lignocellulosic biomass fermentation by the baker's yeast Saccharomyces cerevisiae. Most projects have taken synthetic biological approaches or have explored naturally occurring diversity in S. cerevisiae to enhance stress tolerance, xylose consumption, or ethanol production. Despite these efforts, improved strains with new properties are needed. In other industrial processes, such as wine and beer fermentation, interspecies hybrids have combined important traits from multiple species, suggesting interspecies hybridization may also offer potential for biofuel research. Results: To investigate the efficacy of this approach for traits relevant to lignocellulosic biofuel production, we generated synthetic hybrids by crossing engineered xylose fermenting strains of S. cerevisiae with wild strains from various Saccharomyces species. These interspecies hybrids retained important parental traits, such as xylose consumption and stress tolerance, while displaying intermediate kinetic parameters and, in some cases, heterosis (hybrid vigor). Next, we exposed them to adaptive evolution in Ammonia Fiber Expansion (AFEX)-pretreated Corn Stover Hydrolysate (ACSH) and recovered strains with improved fermentative traits. Genome sequencing showed that the genomes of these evolved synthetic hybrids underwent rearrangements, duplications, and deletions. To determine whether the genus Saccharomyces contains additional untapped potential, we screened a genetically diverse 65 collection of more than 500 wild, non-engineered Saccharomyces isolates and uncovered a wide range of capabilities for traits relevant to cellulosic biofuel production. Notably, Saccharomyces mikatae strains have high innate tolerance to hydrolysate toxins, while some Saccharomyces species have a robust native capacity to consume xylose. Conclusions: This research demonstrates that hybridization is a viable method to combine industrially relevant traits from diverse yeast species and that members of the genus Saccharomyces beyond S. cerevisiae may offer advantageous genes and traits of interest to the lignocellulosic biofuel industry.

Hydroxystilbenes are monomers in palm fruit endocarp lignins

José Carlos Del Rio; Jorge Rencoret; Ana Gutiérrez; Hoon Kim; John Ralph

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2017

Lignin, the plant cell wall polymer that binds fibers together but makes processing difficult, is traditionally formed from three monomers, the so-called monolignols (p-coumaryl, coniferyl, and sinapyl alcohols). Recently we discovered, in grass lignins, a phenolic monomer that falls outside the canonical lignin biosynthetic pathway, the flavone tricin. As we show here, palm fruit (macaúba, carnauba, and coconut) endocarps contain lignin polymers derived in part from a previously unconsidered class of lignin monomers, the hydroxystilbenes, including the valuable compounds piceatannol and resveratrol. Piceatannol could be released from these lignins upon DFRC, a degradative method that cleaves β-ether bonds, indicating that at least a fraction is incorporated through labile ether bonds. NMR spectroscopy of products from the copolymerization of piceatannol and monolignols confirms the structures in the natural polymer, and demonstrates that piceatannol acts as an authentic monomer participating in coupling and cross-coupling reactions during lignification. Palm fruit endocarps therefore contain a new class of 'stilbenolignin' polymers, further expanding the definition of lignin and implying that compounds such as piceatannol and resveratrol are potentially available in what is now essentially a waste product.

Impact of lignin polymer backbone esters on ionic liquid pretreatment of poplar

Kwang Ho Kim; Tanmoy Dutta; John Ralph; Shawn D. Mansfield; Blake A. Simmons; Seema Singh

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2017

Impacts of vegetation type and climatic zone on neutral sugar distribution in natural forest soils

Lefang Cui; Chao Liang; David S. Duncan; Xuelian Bao; Hongtu Xie; Hongbo He; Kyle Wickings; Xudong Zhang; Fusheng Chen

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2017

Soil neutral sugars are a significant component of labile soil organic carbon (SOC) and are derived from both plant and microbial biomass. While plants synthesize both pentose and hexose neutral sugars, microbes almost exclusively produce hexoses. Hexose to pentose ratios in soil thus potentially indicate the extent to which microbes process labile SOC. In this study, we used the ratio of galactose + mannose (G + M) to arabinose + xylose (A + X) to estimate the contribution of sugars derived from microbes and plants to SOC in forest ecosystems. We explored how forest type and climatic zone influence soil neutral sugar profiles by studying coniferous and broadleaf forests located in temperate and subtropical regions in China. At each site, neutral sugars from organic (O) and top-layer mineral (A) soil horizons, as well as from freshly-fallen leaf litter, were measured. Total SOC and soil neutral sugar contents were lower in the subtropical region than in the temperate region, with lower levels in the A horizon than in the O horizon. In both climatic zones, litter (G + M)/(A + X) ratios were higher in coniferous forests (1.2 ± 0.3) than in broadleaf forests (0.4 ± 0.1). Differences in the (G + M)/(A + X) ratios between forest types (coniferous and broadleaf) persisted in the O horizon (1.4 ± 0.2 > 0.9 ± 0.0) and in the A horizon (1.8 ± 0.1 > 1.3 ± 0.0). Across climate zones and forest types, ratios increased from litter over the O horizon to the A horizon. Contrary to our expectations, climate zone did not affect soil (G + M)/(A + X) ratios. Our findings emphasize the important contribution of microbial biomass to labile SOC pools while revealing that soil neutral sugar profiles do not respond to climatic zone drivers as expected.

In Brachypodium a complex signaling is actuated to protect cells from proteotoxic stress and facilitate seed filling

Sang-Jin Kim; Starla Zemelis-Durfee; Curtis Wilkerson; Federica Brandizzi

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2017

MAIN CONCLUSION: A conserved UPR machinery is required for Brachypodium ER stress resistance and grain filling. Human and livestock diets depend on the accumulation of cereal storage proteins and carbohydrates, including mixed-linkage glucan (MLG), in the endosperm during seed development. Storage proteins and proteins responsible for the production of carbohydrates are synthesized in the endoplasmic reticulum (ER). Unfavorable conditions during growth that hamper the ER biosynthetic capacity, such as heat, can cause a potentially lethal condition known as ER stress, which activates the unfolded protein response (UPR), a signaling response designed to mitigate ER stress. The UPR relies primarily on a conserved ER-associated kinase and ribonuclease, IRE1, which splices the mRNA of a transcription factor (TF), such as bZIP60 in plants, to produce an active TF that controls the expression of ER resident chaperones. Here, we investigated activation of the UPR in Brachypodium, as a model to study the UPR in seeds of a monocotyledon species, as well as the consequences of heat stress on MLG deposition in seeds. We identified a Brachypodium bZIP60 orthologue and determined a positive correlation between bZIP60 splicing and ER stress induced by chemicals and heat. Each stress condition led to transcriptional modulation of several BiP genes, supporting the existence of condition-specific BiP regulation. Finally, we found that the UPR is elevated at the early stage of seed development and that MLG production is negatively affected by heat stress via modulation of MLG synthase accumulation. We propose that successful accomplishment of seed filling is strongly correlated with the ability of the plant to sustain ER stress via the UPR.

Increasing the revenue from lignocellulosic biomass: maximizing feedstock utilization

David M. Alonso; Sikander Hakim; Shengfei Zhou; Wangyun Won; Omid Hosseinaei; Jingming Tao; Valerie Garcia-Negron; Ali H. Motagamwala; Max A. Mellmer; Kefeng Huang; Carl J. Houtman; Nicole Labbe; David P. Harper; Christos Maravelias; Troy Runge; James A. Dumesic

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2017

The production of renewable chemicals and biofuels must be cost- and performance- competitive with petroleum-derived equivalents to be widely accepted by markets and society. We propose a biomass conversion strategy that maximizes the conversion of lignocellulosic biomass (up to 80% of the biomass to useful products) into high-value products that can be commercialized, providing the opportunity for successful translation to an economically viable commercial process. Our fractionation method preserves the value of all three primary components: (i) cellulose, which is converted into dissolving pulp for fibers and chemicals production; (ii) hemicellulose, which is converted into furfural (a building block chemical); and (iii) lignin, which is converted into carbon products (carbon foam, fibers, or battery anodes), together producing revenues of more than $500 per dry metric ton of biomass. Once de-risked, our technology can be extended to produce other renewable chemicals and biofuels.

Iron cycling in the anoxic cryo-ecosystem of Antarctic Lake Vida

Bernadette C. Proemse; Alison E. Murray; Christina Schallenberg; Breege McKiernan; Brian T. Glazer; Seth A. Young; Nathaniel E. Ostrom; Andrew R. Bowie; Michael E. Wieser; Fabien Kenig; Peter T. Doran; Ross Edwards

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2017

Iron redox cycling in metal-rich, hypersaline, anoxic brines plays a central role in the biogeochemical evolution of life on Earth, and similar brines with the potential to harbor life are thought to exist elsewhere in the solar system. To investigate iron biogeochemical cycling in a terrestrial analog we determined the iron redox chemistry and isotopic signatures in the cryoencapsulated liquid brines found in frozen Lake Vida, East Antarctica. We used both in situ voltammetry and the spectrophotometric ferrozine method to determine iron speciation in Lake Vida brine (LVBr). Our results show that iron speciation in the anoxic LVBr was, unexpectedly, not free Fe(II). Iron isotope analysis revealed highly depleted values of −2.5‰ for the ferric iron of LVBr that are similar to iron isotopic signatures of Fe(II) produced by dissimilatory iron reduction. The presence of Fe(III) in LVBr therefore indicates dynamic iron redox cycling beyond iron reduction. Furthermore, extremely low δ18O–SO4 2− values (−9.7‰) support microbial iron-sulfur cycling reactions. In combination with evidence for chemodenitrification resulting in iron oxidation, we conclude that coupled abiotic and biotic redox reactions are driving the iron cycle in Lake Vida brine. Our findings challenge the current state of knowledge of anoxic brine chemistry and may serve as an analogue for icy brines found in the outer reaches of the solar system.

Lateral gene transfer dynamics in the ancient bacterial genus Streptomyces

Brandon R. McDonald; Cameron R. Currie

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2017

Lateral gene transfer (LGT) profoundly shapes the evolution of bacterial lineages. LGT across disparate phylogenetic groups and genome content diversity between related organisms suggest a model of bacterial evolution that views LGT as rampant and promiscuous. It has even driven the argument that species concepts and tree-based phylogenetics cannot be applied to bacteria. Here, we show that acquisition and retention of genes through LGT are surprisingly rare in the ubiquitous and biomedically important bacterial genus Streptomyces. Using a molecular clock, we estimate that the Streptomyces bacteria are ~380 million years old, indicating that this bacterial genus is as ancient as land vertebrates. Calibrating LGT rate to this geologic time span, we find that on average only 10 genes per million years were acquired and subsequently maintained. Over that same time span, Streptomyces accumulated thousands of point mutations. By explicitly incorporating evolutionary timescale into our analyses, we provide a dramatically different view on the dynamics of LGT and its impact on bacterial evolution.

Lignin-derived thioacidolysis dimers: reevaluation, new products, authentication, and quantification

Fengxia Yue; Fachuang Lu; Matt Regner; Runcang Sun; John Ralph

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2017

Lignin structural studies play an essential role both in understanding the development of plant cell walls and for valorizing lignocellulosics as renewable biomaterials. Dimeric products released by selectively cleaving β-aryl ether linkages between lignin units reflect the distribution of recalcitrant lignin units, but have been neither absolutely defined nor quantitatively determined. Here we identified and quantified 12 guaiacyl-type thioacidolysis dimers using newly synthesized standards. One product previously attributed to deriving from β–1-coupled units was established as resulting from β– 5 units, correcting an analytical quandary. Another long-standing dilemma, that no β–β dimers were recognized in thioacidolysis products from gymnosperms, has now been resolved with the discovery of two such authenticated compounds. Individual GC response factors for each standard compound allow rigorous quantification of dimeric products released from softwood lignins, affording insight into the various interunit linkage distributions in lignins and thereby guiding the valorization of lignocellulosics.

Lignocellulosic pretreatment in a fungus-cultivating termite

Hongjie Li; Daniel J. Yelle; Chang Li; Mengyi Yang; Jing Ke; Ruijuan Zhang; Yu Liu; Na Zhu; Shiyou Liang; Xiaochang Mo; John Ralph; Cameron R. Currie; Jianchu Mo

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2017

Depolymerizing lignin, the complex phenolic polymer fortifying plant cell walls, is an essential but challenging starting point for the lignocellulosics industries. The variety of ether– and carbon–carbon interunit linkages produced via radical coupling during lignification limit chemical and biological depolymerization efficiency. In an ancient fungus-cultivating termite system, we reveal unprecedentedly rapid lignin depolymerization and degradation by combining laboratory feeding experiments, lignocellulosic compositional measurements, electron microscopy, 2D-NMR, and thermochemolysis. In a gut transit time of under 3.5 h, in young worker termites, poplar lignin sidechains are extensively cleaved and the polymer is significantly depleted, leaving a residue almost completely devoid of various condensed units that are traditionally recognized to be the most recalcitrant. Subsequently, the fungus-comb microbiome preferentially uses xylose and cleaves polysaccharides, thus facilitating final utilization of easily digestible oligosaccharides by old worker termites. This complementary symbiotic pretreatment process in the fungus-growing termite symbiosis reveals a previously unappreciated natural system for efficient lignocellulose degradation.

Maintaining the factory: the roles of the unfolded protein response in cellular homeostasis in plants

Evan Angelos; Cristina Ruberti; Sang-Jin Kim; Federica Brandizzi

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2017

Much like a factory, the endoplasmic reticulum assembles simple cellular building blocks into complex molecular machines known as proteins. In order to protect the delicate protein folding process and ensure the proper cellular delivery of protein products under environmental stresses, eukaryotes have evolved a set of signaling mechanisms known as the unfolded protein response (UPR) to increase the folding capacity of the endoplasmic reticulum. This process is particularly important in plants, because their sessile nature commands adaptation for survival rather than escape from stress. As such, plants make special use of the UPR, and evidence indicates that the master regulators and downstream effectors of the UPR have distinct roles in mediating cellular processes that affect organism growth and development as well as stress responses. In this review we outline recent developments in this field that support a strong relevance of the UPR to many areas of plant life. This article is protected by copyright. All rights reserved.

Major changes in microbial diversity and community composition across gut sections of a juvenile Panchlora cockroach

Erin A. Gontang; Frank O. Aylward; Camila Carlos; Tijana Glavina del Rio; Mansi Chovatia; Alison Fern; Chien-Chi Lo; Stephanie A. Malfatti; Susannah G. Tringe; Cameron R. Currie; Roberto Kolter

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2017

Investigations of gut microbiomes have shed light on the diversity and genetic content of these communities, and helped shape our understanding of how host-associated microorganisms influence host physiology, behavior, and health. Despite the importance of gut microbes to metazoans, our understanding of the changes in diversity and composition across the alimentary tract, and the source of the resident community are limited. Here, using community metagenomics and 16S rRNA gene sequencing, we assess microbial community diversity and coding potential in the foregut, midgut, and hindgut of a juvenile Panchlora cockroach, which resides in the refuse piles of the leaf-cutter ant species Atta colombica. We found a significant shift in the microbial community structure and coding potential throughout the three gut sections of Panchlora sp., and through comparison with previously generated metagenomes of the cockroach’s food source and niche, we reveal that this shift in microbial community composition is influenced by the ecosystems in which Panchlora sp. occurs. While the foregut is composed of microbes that likely originate from the symbiotic fungus gardens of the ants, the midgut and hindgut are composed of a microbial community that is likely cockroach-specific. Analogous to mammalian systems, the midgut and hindgut appear to be dominated by Firmicutes and Bacteroidetes with the capacity for polysaccharide degradation, suggesting they may assist in the degradation of dietary plant material. Our work underscores the prominence of community changes throughout gut microbiomes and highlights ecological factors that underpin the structure and function of the symbiotic microbial communities of metazoans.

Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose

Tam N.T. Tran; Rebecca J. Breuer; Ragothaman A. Narasimhan; Lucas S. Parreiras; Yaoping Zhang; Trey K. Sato; Timothy P. Durrett

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2017

Background: Acetyl-triacylglycerols (acetyl-TAGs) are unusual triacylglycerol (TAG) molecules that contain an sn-3 acetate group. Compared to typical triacylglycerol molecules (here referred to as long chain TAGs; lcTAGs), acetyl-TAGs possess reduced viscosity and improved cold temperature properties, which may allow direct use as a drop-in diesel fuel. Their different chemical and physical properties also make acetyl-TAGs useful for other applications such as lubricants and plasticizers. Acetyl-TAGs can be synthesized by EaDAcT, a diacylglycerol acetyltransferase enzyme originally isolated from Euonymus alatus (Burning Bush). The heterologous expression of EaDAcT in different organisms, including Saccharomyces cerevisiae, resulted in the accumulation of acetyl-TAGs in storage lipids. Microbial conversion of lignocellulose into acetyl-TAGs could allow biorefinery production of versatile molecules for biofuel and bioproducts. Results: In order to produce acetyl-TAGs from abundant lignocellulose feedstocks, we expressed EaDAcT in S. cerevisiae previously engineered to utilize xylose as a carbon source. The resulting strains were capable of producing acetyl-TAGs when grown on different media. The highest levels of acetyl-TAG production were observed with growth on synthetic lab media containing glucose or xylose. Importantly, acetyl-TAGs were also synthesized by this strain in AFEX corn stover hydrolysate (ACSH) at higher volumetric titers than previously published strains. The deletion of the four endogenous enzymes known to contribute to lcTAG production increased the proportion of acetyl-TAGs in the total storage lipids beyond that in existing strains, which will make purification of these useful lipids easier. Surprisingly, the strains containing the four deletions were still capable of synthesizing lcTAG, suggesting that the particular strain used in this study possesses additional undetermined diacylglycerol acyltransferase activity. Interestingly, the carbon source used for growth influenced the accumulation of these residual lcTAGs, with higher levels in strains cultured on xylose containing media. Conclusion: Our results demonstrate that S. cerevisiae can be metabolically engineered to produce acetyl-TAGs when grown on different carbon sources, including hydrolysate derived from lignocellulose. Deletion of four endogenous acyltransferases enabled a higher purity of acetyl-TAGs to be achieved, but lcTAGs were still synthesized. Longer incubation times also decreased the levels of acetyl-TAGs produced. Therefore, additional work is needed to further manipulate acetyl-TAG production in this strain of S. cerevisiae, including the identification of other TAG biosynthetic and lipolytic enzymes and a better understanding of the regulation of the synthesis and degradation of storage lipids.

Metabolic modeling for design of cell factories

Mingyuan Tian; Prashant Kumar; Sanjan T.P. Gupta; Jennifer L. Reed

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2017

There has been a steep rise in the number of genome-scale metabolic models that are available. These models have been a popular choice for designing cell factories given the successes of constraint-based strain design algorithms in metabolic engineering. This chapter provides a description of the various steps involved in building a metabolic model along with tools for facilitating this process. The chapter also describes the major types of constraint-based modeling techniques used for in silico strain design. Case studies are also summarized, illustrating the experimental validation of strain design algorithms and providing practical insights into designing microbial factories.

Mining the isotopic complexity of nitrous oxide: a review of challenges and opportunities

Nathaniel E. Ostrom; Peggy H. Ostrom

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2017

Nitrous oxide (N2O) is an important focus of international greenhouse gas accounting agreements and mitigation of emissions will likely depend on understanding the mechanisms of its formation and reduction. Consequently, applications of stable isotope techniques to understand N2O cycling are proliferating and recent advances in technology are enabling (1) increases in the frequency of isotope analyses and (2) analyses not previously possible. The two isotopes of N and 3 isotopes of O combine to form a total of 12 possible isotopic molecules of N2O. Consequently, this remarkably simple molecule contains a wealth of isotopic information in the form of bulk (δ15N, δ18O), position dependent (site preference), mass independent (Δ17O) and multiply-substituted or clumped isotope compositions. With recent developments in high-mass resolution double sector instruments all 12 isotopic molecules will likely be resolved in the near future. Advances in spectroscopic instruments hold the promise of substantial increases in sample throughput; however, spectroscopic analyses require corrections due to interferences from other gases and frequent and accurate calibration. Mass spectrometric approaches require mass overlap corrections that are not uniform between research groups and interlaboratory comparisons remain imprecise. The continued lack of attention to calibration by both funding agencies and investigators can only perpetuate disagreement between laboratories in reported isotope values for N2O that, in turn, will compromise global assessments of N2O sources and sinks based on isotope ratios. This review discusses the challenges and opportunities offered by the isotopic complexity of N2O.

Mutations that alter the bacterial cell envelope increase lipid production

Kimberly C. Lemmer; Weiping Zhang; Samatha J. Langer; Alice C. Dohnalkova; Dehong Hu; Rachelle A. Lemke; Jeff S. Piotrowski; Galya Orr; Daniel R. Noguera; Timothy J. Donohue

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2017

Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation in Rhodobacter sphaeroides. By screening an R. sphaeroides Tn5 mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their total lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals.

Nannochloropsis, a rich source of diacylglycerol acyltransferases for engineering of triacylglycerol content in different hosts

Krzysztof Zienkiewicz; Agnieszka Zienkiewicz; Eric Poliner; Zhi-Yan Du; Katharina Vollheyde; Cornelia Herrfurth; Sofia Marmon; Eva M. Farre; Ivo Feussner; Christoph Benning

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2017

Background: Photosynthetic microalgae are considered a viable and sustainable resource for biofuel feedstocks, because they can produce higher biomass per land area than plants and can be grown on non-arable land. Among many microalgae considered for biofuel production, Nannochloropsis oceanica (CCMP1779) is particularly promising, because following nutrient deprivation it produces very high amounts of triacylglycerols (TAG). The committed step in TAG synthesis is catalyzed by acyl-CoA: diacylglycerol acyltransferase (DGAT). Remarkably, a total of 13 putative DGAT-encoding genes have been previously identified in CCMP1779 but most have not yet been studied in detail. Results: Based on their expression profile, six out of 12 type-2 DGAT-encoding genes (NoDGTT1-NoDGTT6) were chosen for their possible role in TAG biosynthesis and the respective cDNAs were expressed in a TAG synthesis-deficient mutant of yeast. Yeast expressing NoDGTT5 accumulated TAG to the highest level. Over-expression of NoDGTT5 in CCMP1779 grown in N-replete medium resulted in levels of TAG normally observed only after N deprivation. Reduced growth rates accompanied NoDGTT5 over-expression in CCMP1779. Constitutive expression of NoDGTT5 in Arabidopsis thaliana was accompanied by increased TAG content in seeds and leaves. A broad substrate specificity for NoDGTT5 was revealed, with preference for unsaturated acyl groups. Furthermore, NoDGTT5 was able to successfully rescue the Arabidopsis tag1-1 mutant by restoring the TAG content in seeds. Conclusions: Taken together, our results identified NoDGTT5 as the most promising gene for the engineering of TAG synthesis in multiple hosts among the 13 DGAT-encoding genes of N. oceanica CCMP1779. Consequently, this study demonstrates the potential of NoDGTT5 as a tool for enhancing the energy density in biomass by increasing TAG content in transgenic crops used for biofuel production.

Natural acetylation impacts carbohydrate recovery during deconstruction of Populus trichocarpa wood

Amanda M. Johnson; Hoon Kim; John Ralph; Shawn D. Mansfield

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2017

Background Significant variation in the inherent degree of acetylation naturally exists in the xylem cell walls of Populus trichocarpa. During pretreatment, endogenous acetate hydrolyzes to acetic acid that can subsequently catalyze the breakdown of poplar wood, increasing the efficiency of biomass pretreatment. Results Poplar genotypes varying in cell wall composition were pretreated in 0.3% H2SO4 in non-isothermal batch reactors. Acetic acid released from the wood was positively related to sugar release during pretreatment (R ≥ 0.9), and inversely proportional to the lignin content of the poplar wood (R = 0.6). Conclusion There is significant variation in wood chemistry among P. trichocarpa genotypes. This study elucidated patterns of cell wall deconstruction and clearly links carbohydrate solubilization to acetate release. Tailoring biomass feedstocks for acetate release could enhance pretreatment efficiencies.

New yeasts - new brews: modern approaches to brewing yeast design and development

B. Gibson; J.-M.A. Geertman; C.T. Hittinger; K. Krogerus; D. Libkind; E.J. Louis; F. Magalhães; J.P. Sampaio

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2017

The brewing industry is experiencing a period of change and experimentation largely driven by customer demand for product diversity. This has coincided with a greater appreciation of the role of yeast in determining the character of beer and the widespread availability of powerful tools for yeast research. Genome analysis in particular has helped clarify the processes leading to domestication of brewing yeast and has identified domestication signatures that may be exploited for further yeast development. The functional properties of non-conventional yeast (both Saccharomyces and non-Saccharomyces) are being assessed with a view to creating beers with new flavours as well as producing flavoursome non-alcoholic beers. The discovery of the psychrotolerant S. eubayanus has stimulated research on de novo S. cerevisiae x S. eubayanus hybrids for low-temperature lager brewing and has led to renewed interest in the functional importance of hybrid organisms and the mechanisms that determine hybrid genome function and stability. The greater diversity of yeast that can be applied in brewing, along with an improved understanding of yeasts’ evolutionary history and biology, is expected have a significant and direct impact on the brewing industry, with potential for improved brewing efficiency, product diversity and, above all, customer satisfaction.

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