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2017
Hildebrand, M, Manandhar-Shrestha K, Abbriano R.  2017.  Effects of chrysolaminarin synthase knockdown in the diatom Thalassiosira pseudonana: Implications of reduced carbohydrate storage relative to green algae. Algal Research-Biomass Biofuels and Bioproducts. 23:66-77.   10.1016/j.algal.2017.01.010   AbstractWebsite

In all organisms, the flux of carbon through the fundamental pathways of glycolysis, gluconeogenesis and the pyruvate hub is a core process related to growth and productivity. In unicellular microalgae, the complexity and intracellular location of specific steps of these pathways can vary substantially. In addition, the location and chemical nature of storage carbohydrate can be substantially different. The role of starch storage in green algae has been investigated, but thus far, only a minimal understanding of the role of carbohydrate storage in diatoms as the beta- 1,3-glucan chrysolaminarin has been achieved. In this report, we aimed to determine the effect of specifically reducing the ability of Thalassiosira pseudonana cells to accumulate chrysolaminarin by knocking down transcript levels of the chrysolaminarin synthase gene. We monitored changes in chrysolaminarin and triacylglycerol (TAG) levels during growth and silicon starvation. Transcript- level changes in genes encoding steps in chrysolaminarin metabolism, and cytoplasmic and chloroplast glycolysis/ gluconeogenesis, were monitored during silicon limitation, highlighting the carbon flux processes involved. We demonstrate that knockdown lines accumulate less chrysolaminarin and have a transiently increased TAG level, with minimal detriment to growth. The results provide insight into the role of chrysolaminarin storage in diatoms, and further discussion highlights differences between diatoms and green algae in carbohydrate storage processes and the effect of reducing carbohydrate stores on growth and productivity. (c) 2017 The Authors. Published by Elsevier B.V.

Davis, A, Abbriano R, Smith SR, Hildebrand M.  2017.  Clarification of photorespiratory processes and the role of malic enzyme in diatoms. Protist. 168:134-153.   10.1016/j.protis.2016.10.005   AbstractWebsite

Evidence suggests that diatom photorespiratory metabolism is distinct from other photosynthetic eukaryotes in that there may be at least two routes for the metabolism of the photorespiratory metabolite glycolate. One occurs primarily in the mitochondria and is similar to the C2 photorespiratory pathway, and the other processes glycolate through the peroxisomal glyoxylate cycle. Genomic analysis has identified the presence of key genes required for glycolate oxidation, the glyoxylate cycle, and malate metabolism, however, predictions of intracellular localization can be ambiguous and require verification. This knowledge gap leads to uncertainties surrounding how these individual pathways operate, either together or independently, to process photorespiratory intermediates under different environmental conditions. Here, we combine in silico sequence analysis, in vivo protein localization techniques and gene expression patterns to investigate key enzymes potentially involved in photorespiratory metabolism in the model diatom Thalassiosira pseudonana. We demonstrate the peroxisomal localization of isocitrate lyase and the mitochondrial localization of malic enzyme and a glycolate oxidase. Based on these analyses, we propose an updated model for photorespiratory metabolism in T. pseudonana, as well as a mechanism by which C2 photorespiratory metabolism and its associated pathways may operate during silicon starvation and growth arrest. (C) 2016 Elsevier GmbH. All rights reserved.

Nazmi, A, Hauck R, Davis A, Hildebrand M, Corbeil LB, Gallardo RA.  2017.  Diatoms and diatomaceous earth as novel poultry vaccine adjuvants. Poultry Science. 96:288-294.   10.3382/ps/pew250   AbstractWebsite

Diatoms are single cell eukaryotic microalgae; their surface possesses a porous nanostructured silica cell wall or frustule. Diatomaceous earth (DE) or diatomite is a natural siliceous sediment of diatoms. Since silica has been proved to have adjuvant capabilities, we propose that diatoms and DE may provide an inexpensive and abundant source of adjuvant readily available to use in livestock vaccines. In a first experiment, the safety of diatoms used as an adjuvant for in-ovo vaccination was investigated. In a second experiment, we assessed the humoral immune response after one in-ovo vaccination with inactivated Newcastle Disease Virus (NDV) and DE as adjuvant followed by 2 subcutaneous boosters on d 21 and 29 of age. In both experiments, results were compared to Freund's incomplete adjuvant and aluminum hydroxide. No detrimental effects on hatchability and chick quality were detected after in-ovo inoculation of diatoms and DE in experiments 1 and 2 respectively. In experiment 2 no humoral responses were detected after the in-ovo vaccination until 29 d of age. Seven d after the second subcutaneous booster an antibody response against NDV was detected in chickens that had received vaccines adjuvanted with Freund's incomplete adjuvant, aluminum hydroxide, and DE. These responses became significantly higher 10 d after the second booster. Finally, 15 d after the second booster, the humoral responses induced by the vaccine with Freund's incomplete adjuvant were statistically higher, followed by comparable responses induced by vaccines containing DE or aluminum hydroxide that were significantly higher than DE+PBS, PBS+INDV and PBS alone. From an applied perspective, we can propose that DE can serve as a potential adjuvant for vaccines against poultry diseases.

2016
Traller, JC, Cokus SJ, Lopez DA, Gaidarenko O, Smith SR, McCrow JP, Gallaher SD, Podell S, Thompson M, Cook O, Morselli M, Jaroszewicz A, Allen EE, Allen AE, Merchant SS, Pellegrini M, Hildebrand M.  2016.  Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype. Biotechnology for Biofuels. 9:258.   10.1186/s13068-016-0670-3   AbstractWebsite

Improvement in the performance of eukaryotic microalgae for biofuel and bioproduct production is largely dependent on characterization of metabolic mechanisms within the cell. The marine diatom Cyclotella cryptica, which was originally identified in the Aquatic Species Program, is a promising strain of microalgae for large-scale production of biofuel and bioproducts, such as omega-3 fatty acids.

Cook, O, Hildebrand M.  2016.  Enhancing LC-PUFA production in Thalassiosira pseudonana by overexpressing the endogenous fatty acid elongase genes. Journal of Applied Phycology. 28:897-905.   10.1007/s10811-015-0617-2   AbstractWebsite

The health beneficial omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are naturally synthesized by diatoms through consecutive steps of fatty acid elongase and desaturase enzymes. In Thalassiosira pseudonana, these fatty acids constitute about 10-20 % of the total fatty acids, with EPA accumulation being five to ten times higher than DHA. In order to identify the subcellular localization of enzymes in the pathway of LC-PUFA biosynthesis in T. pseudonana and to manipulate the production of EPA and DHA, we generated constructs for overexpressing each of the T. pseudonana long-chain fatty acid elongase genes. Full-length proteins were fused to GFP, and transgenic lines were generated. In addition, overexpressed native proteins with no GFP fusion were tested. The subcellular localization of each elongase protein was determined. We then examined the total amount of lipids and analyzed the fatty acid profile in each of the transgenic lines compared to wild type. Lines with overexpressed elongases showed an increase of up to 1.4-fold in EPA and up to 4.5-fold in DHA, and the type of fatty acid that was increased (EPA vs. DHA) depended on the type of elongase that was overexpressed. This data informs future metabolic engineering approaches to further improve EPA and DHA content in diatoms.

Beld, J, Abbriano R, Finzel K, Hildebrand M, Burkart MD.  2016.  Probing fatty acid metabolism in bacteria, cyanobacteria, green microalgae and diatoms with natural and unnatural fatty acids. Molecular Biosystems. 12:1299-1312.   10.1039/c5mb00804b   AbstractWebsite

In both eukaryotes and prokaryotes, fatty acid synthases are responsible for the biosynthesis of fatty acids in an iterative process, extending the fatty acid by two carbon units every cycle. Thus, odd numbered fatty acids are rarely found in nature. We tested whether representatives of diverse microbial phyla have the ability to incorporate odd-chain fatty acids as substrates for their fatty acid synthases and their downstream enzymes. We fed various odd and short chain fatty acids to the bacterium Escherichia coli, cyanobacterium Synechocystis sp. PCC 6803, green microalga Chlamydomonas reinhardtii and diatom Thalassiosira pseudonana. Major differences were observed, specifically in the ability among species to incorporate and elongate short chain fatty acids. We demonstrate that E. coli, C. reinhardtii, and T. pseudonana can produce longer fatty acid products from short chain precursors (C3 and C5), while Synechocystis sp. PCC 6803 lacks this ability. However, Synechocystis can incorporate and elongate longer chain fatty acids due to acyl-acyl carrier protein synthetase (AasS) activity, and knockout of this protein eliminates the ability to incorporate these fatty acids. In addition, expression of a characterized AasS from Vibrio harveyii confers a similar capability to E. coli. The ability to desaturate exogenously added fatty acids was only observed in Synechocystis and C. reinhardtii. We further probed fatty acid metabolism of these organisms by feeding desaturase inhibitors to test the specificity of long-chain fatty acid desaturases. In particular, supplementation with thia fatty acids can alter fatty acid profiles based on the location of the sulfur in the chain. We show that coupling sensitive gas chromatography mass spectrometry to supplementation of unnatural fatty acids can reveal major differences between fatty acid metabolism in various organisms. Often unnatural fatty acids have antibacterial or even therapeutic properties. Feeding of short precursors now gives us easy access to these extended molecules.

Smith, SR, Glé C, Abbriano RM, Traller JC, Davis A, Trentacoste E, Vernet M, Allen AE, Hildebrand M.  2016.  Transcript level coordination of carbon pathways during silicon starvation-induced lipid accumulation in the diatom Thalassiosira pseudonana. New Phytologist.   10.1111/nph.13843   Abstract

* Diatoms are one of the most productive and successful photosynthetic taxa on Earth and possess attributes such as rapid growth rates and production of lipids, making them candidate sources of renewable fuels. Despite their significance, few details of the mechanisms used to regulate growth and carbon metabolism are currently known, hindering metabolic engineering approaches to enhance productivity. * To characterize the transcript level component of metabolic regulation, genome-wide changes in transcript abundance were documented in the model diatom Thalassiosira pseudonana on a time-course of silicon starvation. Growth, cell cycle progression, chloroplast replication, fatty acid composition, pigmentation, and photosynthetic parameters were characterized alongside lipid accumulation. * Extensive coordination of large suites of genes was observed, highlighting the existence of clusters of coregulated genes as a key feature of global gene regulation in T. pseudonana. The identity of key enzymes for carbon metabolic pathway inputs (photosynthesis) and outputs (growth and storage) reveals these clusters are organized to synchronize these processes. * Coordinated transcript level responses to silicon starvation are probably driven by signals linked to cell cycle progression and shifts in photophysiology. A mechanistic understanding of how this is accomplished will aid efforts to engineer metabolism for development of algal-derived biofuels.

Hildebrand, M, Davis A, Abbriano R, Pugsley HR, Traller JC, Smith SR, Shrestha RP, Cook O, Sanchez E, Manabe S, Manandhar-Shrestha K, Alderete B.  2016.  Applications of Imaging Flow Cytometry for Microalgae. Imaging flow cytometry : methods and protocols. ( Barteneva NS, Vorobjev IA, Eds.).:xiii,295pages.: Humana Press Abstract
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2015
Manandhar-Shrestha, K, Hildebrand M.  2015.  Characterization and manipulation of a DGAT2 from the diatom Thalassiosira pseudonana: Improved TAG accumulation without detriment to growth, and implications for chloroplast TAG accumulation. Algal Research-Biomass Biofuels and Bioproducts. 12:239-248.   10.1016/j.algal.2015.09.004   AbstractWebsite

Improving the ability ofmicroalgae cells to accumulate triacylglycerol (TAG) without a negative effect on growth or biomass accumulation is desirable for economically-viable biofuels production. Metabolic engineering is one method to increase TAG yields, provided that appropriate gene target(s) are identified. Microalgal lipid and TAG synthesis is incompletely characterized, but it is clear that it differs in fundamental ways from plants and other eukaryotes. We characterize a DGAT2 homolog from the diatom Thalassiosira pseudonana, and show that it shifts intracellular location from the chloroplast during exponential growth, to the endoplasmic reticulum in stationary phase when large cytoplasmic lipid droplets accumulate. This is the first direct localization of a chloroplast DGAT2, which confirms the ability of these organelles to accumulate TAG in evolutionarily-diverse classes of microalgae. The enzyme is also not directly localized to cytoplasmic lipid droplets, suggesting that TAG synthesis and lipid droplet formation are separate processes. Overexpression of the enzyme resulted in significantly increased TAG accumulation without a negative effect on growth or biomass accumulation. Overexpression lines had a distinct shift in fatty acid composition relative to wild-type. The results shed light on fundamental processes of algal lipid metabolism and a practical benefit of improved productivity in transgenic lines. (C) 2015 Elsevier B. V. All rights reserved.

Hildebrand, M, Lerch SJL.  2015.  Diatom silica biomineralization: Parallel development of approaches and understanding. Seminars in Cell & Developmental Biology. 46:27-35.   10.1016/j.semcdb.2015.06.007   AbstractWebsite

Diatom silica cell walls present an intriguing application of biomineralization in a single celled organism. The ability of diatoms to make an enormous variety of silica structures on the nano- to micro-scale is unparalleled in nature. The process is a whole-cell endeavor, involving diverse cellular components that coordinate "bottom up" and "top down" structure formation processes to reproducibly convert genetic information into physical structure. The study of silicification has been similarly all encompassing, involving the application of diverse analytical techniques to examine different aspects of the process. This review highlights the application of different approaches used to study silicification and the insights they have provided, and documents the progress that has been made. The current status offers the possibility of major breakthroughs in our understanding, by enabling a more widespread identification of genes involved, and direct testing of the role these genes play by genetic manipulation. (C) 2015 Elsevier Ltd. All rights reserved.

Shrestha, RP, Hildebrand M.  2015.  Evidence for a regulatory role of diatom silicon transporters in cellular silicon responses. Eukaryotic Cell. 14:29-40.   10.1128/ec.00209-14   AbstractWebsite

The utilization of silicon by diatoms has both global and small-scale implications, from oceanic primary productivity to nano-technological applications of their silica cell walls. The sensing and transport of silicic acid are key aspects of understanding diatom silicon utilization. At low silicic acid concentrations (<30 mu M), transport mainly occurs through silicic acid transport proteins (SITs), and at higher concentrations it occurs through diffusion. Previous analyses of the SITs were done either in heterologous systems or without a distinction between individual SITs. In the present study, we examined individual SITs in Thalassiosira pseudonana in terms of transcript and protein abundance in response to different silicic acid regimes and examined knockdown lines to evaluate the role of the SITs in transport, silica incorporation, and lipid accumulation resulting from silicon starvation. SIT1 and SIT2 were localized in the plasma membrane, and protein levels were generally inversely correlated with cellular silicon needs, with a distinct response being found when the two SITs were compared. We developed highly effective approaches for RNA interference and antisense knockdowns, the first such approaches developed for a centric diatom. SIT knockdown differentially affected the uptake of silicon and the incorporation of silicic acid and resulted in the induction of lipid accumulation under silicon starvation conditions far earlier than in the wild-type cells, suggesting that the cells were artificially sensing silicon limitation. The data suggest that the transport role of the SITs is relatively minor under conditions with sufficient silicic acid. Their primary role is to sense silicic acid levels to evaluate whether the cell can proceed with its cell wall formation and division processes.

2014
Pan, ZW, Lerch SJL, Xu L, Li XF, Chuang YJ, Howe JY, Mahurin SM, Dai S, Hildebrand M.  2014.  Electronically transparent graphene replicas of diatoms: a new technique for the investigation of frustule morphology. Scientific Reports. 4   10.1038/srep06117   AbstractWebsite

The morphogenesis of the silica cell walls (called frustules) of unicellular algae known as diatoms is one of the most intriguing mysteries of the diatoms. To study frustule morphogenesis, optical, electron and atomic force microscopy has been extensively used to reveal the frustule morphology. However, since silica frustules are opaque, past observations were limited to outer and fracture surfaces, restricting observations of interior structures. Here we show that opaque silica frustules can be converted into electronically transparent graphene replicas, fabricated using chemical vapor deposition of methane. Chemical vapor deposition creates a continuous graphene coating preserving the frustule's shape and fine, complicated internal features. Subsequent dissolution of the silica with hydrofluoric acid yields a free-standing replica of the internal and external native frustule morphologies. Electron microscopy renders these graphene replicas highly transparent, revealing previously unobserved, complex, three-dimensional, interior frustule structures, which lend new insights into the investigation of frustule morphogenesis.

Kieu, K, Li C, Fang Y, Cohoon G, Herrera OD, Hildebrand M, Sandhage KH, Norwood RA.  2014.  Structure-based optical filtering by the silica microshell of the centric marine diatom Coscinodiscus wailesii. Optics Express. 22:15992-15999.   10.1364/oe.22.015992   AbstractWebsite

Diatoms are a renewable (biologically reproducible) source of three-dimensional (3-D) nanostructured silica that could be attractive for a variety of photonic devices, owing to the wide range of quasi-periodic patterns of nano-to-microscale pores available on the silica microshells (frustules) of various diatom species. We have investigated the optical behavior of the silica frustule of a centric marine diatom, Coscinodiscus wailesii, using a coherent broadband (400-1700 nm) supercontinuum laser focused to a fine (20 mu m diameter) spot. The C. wailesii frustule valve, which possessed a quasi-periodic hexagonal pore array, exhibited position-dependent optical diffraction. Changes in such diffraction behavior across the frustule were consistent with observed variations in the quasi-periodic pore pattern. (C)2014 Optical Society of America

Kopanska, KS, Tesson B, Lin HS, Meredith JC, Hildebrand M, Davis A.  2014.  Morphological factors involved in adhesion of acid-cleaned diatom silica. Silicon. 6:95-107.   10.1007/s12633-014-9178-2   AbstractWebsite

Purpose Diatoms, unicellular microalgae with silica cell walls, have strong adhesive properties, which are dominated by chemical interactions between secreted organic material and the substrate. Possible technological applications of diatoms are likely to involve the adhesion of silica particles, or derivatives, which have been cleaned of organic material. Because the morphologies of diatom cell walls are far more complex than defined model structures, the relationship between morphology and adhesion for such materials is unknown. Methods In this paper we develop a new approach to monitor the adhesion of acid-cleaned diatom silica using parallelplate flow chambers. We have evaluated factors such as settling time, extent of dryness, and substrate properties, and compared diatom species with silica features differing in size, shape, and percentage of surface contact area. Results Results indicated better adhesion of particles with higher surface contact area below a threshold of overall size, and a contribution by the number of possible contact surfaces to initial adhesion. We identified two stages in adhesion response to increasing shear stress. In the first stage, at low shear stress, species-dependent morphology played a major role in determining the strength of adhesion. After loosely adhered particles were removed at low shear, a second stage of persistent adhesion emerged at higher shear stresses. In the second stage, variations in morphology had a much smaller effect on adhesion. Conclusions These results identify conditions and fundamental morphological features for adhesion that can be utilized in future technological applications of silica particles with complex shapes.

2013
Traller, JC, Hildebrand M.  2013.  High throughput imaging to the diatom Cyclotella cryptica demonstrates substantial cell-to-cell variability in the rate and extent of triacylglycerol accumulation. Algal Research-Biomass Biofuels and Bioproducts. 2:244-252.   10.1016/j.algal.2013.03.003   AbstractWebsite

In microalgal cultures, most analyses of cellular processes are done in bulk, on the entire population of cells. Information gained from this is representative of the mean; however, it obscures the richness of cell-to-cell variation, which is a well-documented phenomenon. Using imaging flow cytometry, we evaluate changes in triacylglycerol (TAG) content and chlorophyll resulting from silicon or nitrogen deprivation in the diatom Cyclotella cryptica. This approach allows detailed interrogation of large numbers of individual cells and reveals cell-to-cell variation. This study demonstrates several previously undescribed phenomena related to TAG accumulation in microalgae. First, the rate of TAG accumulation varies over time, with a faster rate occurring at the latter stage of the process, resulting in hyperaccumulation in which the majority of the cell volume is comprised of lipid droplets. In C. cryptica and other diatoms this hyperaccumulation occurs strictly under autotrophic conditions. Second, there are distinct responses to silicon or nitrogen limitation, and variation within a given type of limitation treatment. Under most conditions there is a large spread in the population when measuring either chlorophyll or TAG. Heterogeneity within the total population indicates that caution should be taken in interpreting bulk measurements for a variety of variables (TAG, transcript, protein, metabolites, etc.) related to cellular responses. However, a potential means to couple subpopulation-level responses with bulk analysis approaches is described, which could take advantage of the nuances observed during the TAG accumulation process. (C) 2013 Elsevier B. V. All rights reserved.

Hildebrand, M, Abbriano RM, Polle JE, Traller JC, Trentacoste EM, Smith SR, Davis AK.  2013.  Metabolic and cellular organization in evolutionarily diverse microalgae as related to biofuels production. Current Opinion in Chemical Biology. 17:506-514.   10.1016/j.cbpa.2013.02.027   AbstractWebsite

Microalgae are among the most diverse organisms on the planet, and as a result of symbioses and evolutionary selection, the configuration of core metabolic networks is highly varied across distinct algal classes. The differences in photosynthesis, carbon fixation and processing, carbon storage, and the compartmentation of cellular and metabolic processes are substantial and likely to transcend into the efficiency of various steps involved in biofuel molecule production. By highlighting these differences, we hope to provide a framework for comparative analyses to determine the efficiency of the different arrangements or processes. This sets the stage for optimization on the based on information derived from evolutionary selection to diverse algal classes and to synthetic systems.

Tesson, B, Hildebrand M.  2013.  Characterization and Localization of Insoluble Organic Matrices Associated with Diatom Cell Walls: Insight into Their Roles during Cell Wall Formation. Plos One. 8   10.1371/journal.pone.0061675   AbstractWebsite

Organic components associated with diatom cell wall silica are important for the formation, integrity, and function of the cell wall. Polysaccharides are associated with the silica, however their localization, structure, and function remain poorly understood. We used imaging and biochemical approaches to describe in detail characteristics of insoluble organic components associated with the cell wall in 5 different diatom species. Results show that an insoluble organic matrix enriched in mannose, likely the diatotepum, is localized on the proximal surface of the silica cell wall. We did not identify any organic matrix embedded within the silica. We also identified a distinct material consisting of glucose polymer with variable localization depending on the species. In some species this component was directly involved in the morphogenesis of silica structure while in others it appeared to be only a structural component of the cell wall. A novel glucose-rich structure located between daughter cells during division was also identified. This work for the first time correlates the structure, composition, and localization of insoluble organic matrices associated with diatom cell walls. Additionally we identified a novel glucose polymer and characterized its role during silica structure formation.

Trentacoste, EM, Shrestha RP, Smith SR, Gle C, Hartmann AC, Hildebrand M, Gerwick WH.  2013.  Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth. Proceedings of the National Academy of Sciences of the United States of America. 110:19748-19753.   10.1073/pnas.1309299110   AbstractWebsite

Biologically derived fuels are viable alternatives to traditional fossil fuels, and microalgae are a particularly promising source, but improvements are required throughout the production process to increase productivity and reduce cost. Metabolic engineering to increase yields of biofuel-relevant lipids in these organisms without compromising growth is an important aspect of advancing economic feasibility. We report that the targeted knockdown of a multifunctional lipase/phospholipase/acyltransferase increased lipid yields without affecting growth in the diatom Thalassiosira pseudonana. Antisense-expressing knockdown strains 1A6 and 1B1 exhibited wild-type-like growth and increased lipid content under both continuous light and alternating light/dark conditions. Strains 1A6 and 1B1, respectively, contained 2.4- and 3.3-fold higher lipid content than wild-type during exponential growth, and 4.1- and 3.2-fold higher lipid content than wild-type after 40 h of silicon starvation. Analyses of fatty acids, lipid classes, and membrane stability in the transgenic strains suggest a role for this enzyme in membrane lipid turnover and lipid homeostasis. These results demonstrate that targeted metabolic manipulations can be used to increase lipid accumulation in eukaryotic microalgae without compromising growth.

Manandhar-Shrestha, HKM.  2013.  Development of flow cytometric procedures for the highly efficient isolation of improved lipid accumulation mutants in a Chlorella–like microalga. J. Appl. Phycol..   DOI 10.1007/s10811-013-0021-8   Abstract

The successful development of microalgae-based biofuel production will rely on improvements in the amount and rate of fuel molecule precursor accumulation. Mutagenesis and selection is an attractive approach to improve fuel molecule productivity. Previous screening methods have been laborious, the numbers of mutants isolated have been small, and overall performance of mutants may have been compromised by the presence of deleterious secondary mutations generated by random mutagenesis that affect other cellular processes and growth. We report an improved method of isolating high triacylglycerol (TAG) accumulating mutants of a Chlorella sp., KAS603, using flow cytometric-based selection. In addition to selection for high TAG accumulating strains, the method requires that growth of mutants be competitive with other cells in the population. Not only is growth competitive, but there is improved performance in TAG accumulation with repeated selection, suggesting purification from deleterious secondary mutations. The procedure resulted in the isolation of mutants with far higher efficiency (thousands of fold) that outperformed wild type substantially better (1.8–2.5-fold) than with previous methods. This opens the door to new approaches to the characterization of genes involved in TAG accumulation and other cellular processes.

2012
Shrestha, RP, Tesson B, Norden-Krichmar T, Federowicz S, Hildebrand M, Allen AE.  2012.  Whole transcriptome analysis of the silicon response of the diatom Thalassiosira pseudonana. BMC Genomics. 13:499.   doi:10.1186/1471-2164-13-499   Abstract

Background: Silicon plays important biological roles, but the mechanisms of cellular responses to silicon are poorly understood. We report the first analysis of cell cycle arrest and recovery from silicon starvation in the diatom Thalassiosira pseudonana using whole genome microarrays. Results: Three known responses to silicon were examined, 1) silicified cell wall synthesis, 2) recovery from silicon starvation, and 3) co-regulation with silicon transporter (SIT) genes. In terms of diatom cell wall formation, thus far only cell surface proteins and proteins tightly associated with silica have been characterized. Our analysis has identified new genes potentially involved in silica formation, and other genes potentially involved in signaling, trafficking, protein degradation, glycosylation and transport, which provides a larger-scale picture of the processes involved. During silicon starvation, an overrepresentation of transcription and translation related genes were up-regulated, indicating that T. pseudonana is poised to rapidly recover from silicon starvation and resume cell cycle progression upon silicon replenishment. This is in contrast to other types of limitation, and provides the first molecular data explaining the well-established environmental response of diatoms to grow as blooms and to out-compete other classes of microalgae for growth. Comparison of our data with a previous diatom cell cycle analysis indicates that assignment of the cell cycle specific stage of particular cyclins and cyclin dependent kinases should be re-evaluated. Finally, genes co-varying in expression with the SITs enabled identification of a new class of diatom-specific proteins containing a unique domain, and a putative silicon efflux protein. Conclusions: Analysis of the T. pseudonana microarray data has provided a wealth of new genes to investigate previously uncharacterized cellular phenomenon related to silicon metabolism, silicon's interaction with cellular components, and environmental responses to silicon.

Curnow, P, Senior L, Knight MJ, Thamatrakoln K, Hildebrand M, Booth PJ.  2012.  Expression, Purification, and Reconstitution of a Diatom Silicon Transporter. Biochemistry. 51:3776-3785.   10.1021/bi3000484   AbstractWebsite

The synthesis and manipulation of silicon materials on mineralization increasingly being taken from the natural world because the the nanoscale are core themes in nanotechnology research. Inspiration nanometer. One fascinating example of silicon biomineralization complex silica structures with dimensions from the millimeter to the biological mineralization of silicon results in precisely controlled, occurs in the diatoms, unicellular algae that sheath themselves in an ornate silica-based cell wall. To harvest silicon from the environment, diatoms have developed a unique family of integral membrane proteins that bind to a soluble form of silica, silicic acid, and transport it across the cell membrane to the cell interior. These are the first proteins shown to directly interact with silicon, but the current understanding of these specific silicon transport proteins is limited by the lack of in vitro studies of structure and function. We report here the recombinant expression, purification, and reconstitution of a silicon transporter from the model diatom Thalassiosira pseudonana. After using GFP fusions to optimize expression and purification protocols, a His(10)-tagged construct was expressed in Saccharomyces cerevisiae, solubilized in the detergent Fos-choline-12, and purified by affinity chromatography. Size-exclusion chromatography and particle sizing by dynamic light scattering showed that the protein was purified as a homotetramer, although nonspecific oligomerization occurred at high protein concentrations. Circular dichroism measurements confirmed sequence-based predictions that silicon transporters are a-helical membrane proteins. Silicic acid transport could be established in reconstituted proteoliposomes, and silicon uptake was found to be dependent upon an applied sodium gradient. Transport data across different substrate concentrations were best fit to the sigmoidal Hill equation, with a K-0.5 of 19.4 +/- 1.3 mu M and a cooperativity coefficient of 1.6. Sodium binding was noncooperative with a K-m(aPP) of 1.7 +/- 1.0 mM, suggesting a transport silicic acid:Na+ stoichiometry of 2:1. These results provide the basis for a full understanding of both silicon transport in the diatom and protein silicon interactions in general.

Hildebrand M, Davis AK, SSRTJCAR.  2012.  The place of diatoms in the biofuels industry. Biofuels. 3(2):221–240.   10.4155/BFS.11.157  
Smith, SR, Abbriano RM, Hildebrand M.  2012.  Comparative analysis of diatom genomes reveals substantial differences in the organization of carbon partitioning pathways. Algal Research. 1:2-16.   http://dx.doi.org/10.1016/j.algal.2012.04.003   AbstractWebsite

A major challenge in the development of microalgal strains for large-scale production is the optimization of biomass accumulation and production of fuel-relevant molecules such as triacylglycerol. Selecting targets for genetic manipulation approaches will require a fundamental understanding of the organization and regulation of carbon metabolic pathways in these organisms. Functional genomic and metabolomics data is becoming easier to obtain and process, however interpreting the significance of these data in a physiological context is challenging since the metabolic framework of all microalgae remains poorly understood. Owing to a complex evolutionary history, diatoms differ substantially from many other photosynthetic organisms in their intracellular compartmentation and the organization of their carbon partitioning pathways. A comparative analysis of the genes involved in carbon partitioning metabolism from Thalassiosira pseudonana, Phaeodactylum tricornutum, and Fragilariopsis cylindrus revealed that diatoms have conserved the lower half of glycolysis in the mitochondria, the upper half of glycolysis (including key regulatory enzymes) in the cytosol, and several mitochondrial carbon partitioning enzymes. However, some substantial differences exist between the three diatoms investigated, including the translocation of metabolic pathways to different compartments, selective maintenance and horizontal acquisition of genes, and differential gene family expansions. A key finding is that metabolite transport between intracellular compartments is likely to play a substantial role in the regulation of carbon flux. Analysis of the carbon partitioning components in the mitochondria suggests an important role of this organelle as a carbon flux regulator in diatoms. Differences between the analyzed species are specific examples of how diatoms may have modified their carbon partitioning pathways to adapt to environmental niches during the diversification of the group. This comparative analysis highlights how even core central pathways can be modified considerably within a single algal group, and enables the identification of suitable targets for genetic engineering to enhance biofuel precursor production.

2011
Norden-Krichmar, TM, Allen AE, Gaasterland T, Hildebrand M.  2011.  Characterization of the Small RNA Transcriptome of the Diatom, Thalassiosira pseudonana. Plos One. 6(8):e22870.   10.1371/journal.pone.0022870   AbstractWebsite

This study presents the first characterization of endogenous small RNAs in a diatom, Thalassiosira pseudonana. Small RNAs act as transcriptional and translational regulators, controlling specific target genes involved in various cellular functions. Diatoms are unicellular photosynthetic organisms that play major roles in environmental processes, such as food webs and global carbon fixation. Small RNA cDNA libraries were constructed for exponentially growing T. pseudonana, and then subjected to highly parallel pyrosequencing (454) and sequencing-by-ligation (Applied Biosystems SOLiD). From the computational analysis of approximately 300,000 sequences in the 454 library and over 17 million sequences in the SOLiD libraries, there exists evidence of a core set of small RNA genes including: novel microRNAs, repeat-associated short interfering RNAs, and endogenous short interfering RNAs. The diatom genome contains elements similar to plant small RNA systems, such as the RNAi machinery, a high percentage of short interfering RNAs originating from protein-coding and repetitive regions of the genome, and putative binding sites of the small RNAs occurring primarily in the coding section of the predicted targets. The characterization of the small RNA transcriptome of T. pseudonana establishes the possibility of a wide range of gene regulatory mechanisms in diatoms.

2010
Tesson, B, Hildebrand M.  2010.  Dynamics of silica cell wall morphogenesis in the diatom Cyclotella cryptica: Substructure formation and the role of microfilaments. Journal of Structural Biology. 169:62-74.   10.1016/j.jsb.2009.08.013   AbstractWebsite

Diatoms are unicellular algae that make cell walls out of silica with highly ornate features on the nano- to microscale. The complexity and variety of diatom cell wall structures exceeds those possible with synthetic materials chemistry approaches. Understanding the design and assembly processes involved in diatom silicification should provide insight into patterning on the unicellular level, and information for biomimetic approaches for materials synthesis. In this report we examine the formation of distinct cell wall structures (valves and girdle bands) in the diatom Cyclotella cryptica by high resolution imaging using SEM, AFM, and fluorescence microscopy. Special attention was paid to imaging structural intermediates, which provided insight into the underlying design and assembly principles involved. Distinct stages in valve formation were identified, indicating a transition from a fractally organized structure to a dynamic pathway-dependent process. Substructures in the valves appeared to be pre-positioned prior to complete silicification, suggesting that organics responsible for these structures were pre-assembled and put in place. Microtubules and microfilamentous actin play significant roles in the positioning process, and actin is also important in the pathway-dependent expansion of the front of silicification. Our results indicate that even though all silica structures in C. cryptica are made of assemblies of nanoparticulate silica, control of meso- and microscale structure occurs on a higher order. It is apparent that diatoms integrate bottom up and top down control and synthesis mechanisms to form the diversity of structures possible. (C) 2009 Elsevier Inc. All rights reserved.