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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Carbon source-induced changes in the transcriptional landscape of Novacetimonas hansenii

Novacetimonas hansenii is a model organism for bacterial cellulose (BC) production, a biopolymer with broad industrial applications. To better understand its carbon source-dependent metabolism, we conducted the first time-resolved transcriptomic analysis of N. hansenii ATCC 23769 grown on five carbon sources (glucose, fructose, mannitol, galactose, and sucrose) across five time points (24–120h). Glucose, fructose, and mannitol supported growth and efficient BC production, whereas galactose and sucrose resulted in poor yields, likely due to limited transport and enzymatic capacity. Solid-state NMR revealed that cellulose crystallinity and polymorphism were carbon source-dependent, with reduced bcsD expression correlating with lower crystallinity. Transcriptomic profiling showed distinct metabolic responses. Cultures containing glucose and mannitol activated core metabolic pathways, fructose induced a delayed adaptive shift, and galactose and sucrose triggered stress-related gene expression. These results highlight the metabolic plasticity of N. hansenii and offer insights for enhancing BC production through carbon source optomization.

Bacterial cellulose↗
DOE OSTI2021

The evolution of the GAL actose utilization pathway in budding yeasts

The Leloir galactose utilization or GAL pathway of budding yeasts, including that of the baker’s yeast Saccharomyces cerevisiae and the opportunistic human pathogen Candida albicans, breaks down the sugar galactose for energy and biomass production. Here, the GAL pathway has long served as a model system for understanding how eukaryotic metabolic pathways, including their modes of regulation, evolve. More recently, the physical linkage of the structural genes GAL1, GAL7, and GAL10 in diverse budding yeast genomes has been used as a model for understanding the evolution of gene clustering. In this review, we summarize exciting recent work on three different aspects of this iconic pathway’s evolution: gene cluster organization, GAL gene regulation, and the population genetics of the GAL pathway.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Enhancing the Solubility of Semiconducting Polymers in Eco-Friendly Solvents with Carbohydrate-Containing Side Chains

Semiconducting polymers are at the forefront of next-generation organic electronics due to their robust mechanical and optoelectronic properties. However, their extended π-conjugation often leads to materials with low solubilities in common organic solvents, thus requiring processing in high-boiling-point and toxic halogenated solvents to generate thin-film devices. To address this environmental concern, a natural product-inspired side-chain engineering approach was used to incorporate galactose-containing moieties into semiconducting polymers toward improved processability in greener solvents. Novel isoindigo-based polymers with different ratios of galactose-containing side chains were synthesized to improve the solubilities of the organic semiconductors in alcohol-based solvents. The addition of carbohydrate-containing side chains to π-conjugated polymers was found to considerably impact the intermolecular aggregation of the materials and their microstructures in the solid state as confirmed by atomic force microscopy and grazing-incidence wide-angle X-ray scattering. The charge transport characteristics of the new semiconductors were evaluated by the fabrication of organic field-effect transistors prepared from both toxic halogenated and greener alcohol-based solvents. Importantly, the incorporation of carbohydrate-containing side chains was shown to have very little detrimental impact on the electronic properties of the polymer when processed from green solvents.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Galactomannan utilization by Cellvibrio japonicus relies on a single essential α‐galactosidase encoded by the aga27A gene

Plant mannans are a component of lignocellulose that can have diverse compositions in terms of its backbone and side-chain substitutions. Consequently, the degradation of mannan substrates requires a cadre of enzymes for complete reduction to substituent monosaccharides that can include mannose, galactose, and/or glucose. One bacterium that possesses this suite of enzymes is the Gram-negative saprophyte Cellvibrio japonicus, which has 10 predicted mannanases from the Glycoside Hydrolase (GH) families 5, 26, and 27. Here we describe a systems biology approach to identify and characterize the essential mannan-degrading components in this bacterium. The transcriptomic analysis uncovered significant changes in gene expression for most mannanases, as well as many genes that encode carbohydrate active enzymes (CAZymes) when mannan was actively being degraded. A comprehensive mutational analysis characterized 54 CAZyme-encoding genes in the context of mannan utilization. Growth analysis of the mutant strains found that the man26C, aga27A, and man5D genes, which encode a mannobiohydrolase, α-galactosidase, and mannosidase, respectively, were important for the deconstruction of galactomannan, with Aga27A being essential. Our updated model of mannan degradation in C. japonicus proposes that the removal of galactose sidechains from substituted mannans constitutes a crucial step for the complete degradation of this hemicellulose.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Characterization of the Neurospora crassa Galactosaminogalactan Biosynthetic Pathway

The Neurospora crassa genome has a gene cluster for the synthesis of galactosaminogalactan (GAG). The gene cluster includes the following: (1) UDP-glucose-4-epimerase to convert UDP-glucose and UDP-N-acetylglucosamine to UDP-galactose and UDP-N-acetylgalactosamine (NCU05133), (2) GAG synthase for the synthesis of an acetylated GAG (NCU05132), (3) GAG deacetylase (/NCW-1/NCU05137), (4) GH135-1, a GAG hydrolase with specificity for N-acetylgalactosamine-containing GAG (NCU05135), and (5) GH114-1, a galactosaminidase with specificity for galactosamine-containing GAG (NCU05136). The deacetylase was previously shown to be a major cell wall glycoprotein and given the name of NCW-1 (non-GPI anchored cell wall protein-1). Characterization of the polysaccharides found in the growth medium from the wild type and the GAG synthase mutant demonstrates that there is a major reduction in the levels of polysaccharides containing galactosamine and N-acetylgalactosamine in the mutant growth medium, providing evidence that the synthase is responsible for the production of a GAG. The analysis also indicates that there are other galactose-containing polysaccharides produced by the fungus. Phenotypic characterization of wild-type and mutant isolates showed that deacetylated GAG from the wild type can function as an adhesin to a glass surface and provides the fungal mat with tensile strength, demonstrating that the deacetylated GAG functions as an intercellular adhesive. The acetylated GAG produced by the deacetylase mutant was found to function as an adhesive for chitin, alumina, celite (diatomaceous earth), activated charcoal, and wheat leaf particulates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Broccoli seed extract rich in polysaccharides and glucoraphanin ameliorates DSS ‐induced colitis via intestinal barrier protection and gut microbiota modulation in mice

Abstract BACKGROUND Broccoli has received widespread attention because of its anti‐inflammatory and antioxidant effects. The present study aimed to explore the composition of broccoli seed extract (BSE) and its effect on colitis induced by dextran sulfate sodium (DSS). RESULTS BSE mainly comprises glucoraphanin and polysaccharides composed of arabinose, galactose, glucose and mannose. Animal experiments suggested that BSE intervention effectively reversed body weight loss, suppressed the levels of proinflammatory interleukin‐6, tumor necrosis factor‐α and interleukin‐1β, and elevated the levels of anti‐inflammatory interleukin‐10 and the activities of superoxide dismutase and glutathione in DSS‐induced colitis mice. According to histopathologic and immunohistochemical analysis of colon tissue, BSE intervention may repair the intestinal barrier by upregulating mRNA levels and the expression of tight junction proteins (claudin‐1, occludin and zonula occludens‐1). Gas chromatography–mass spectrometry (MS) analysis demonstrated that cecal short‐chain fatty acids in mice with BSE administration were significantly increased compared with the model group. Sulforaphane and sulforaphane‐ N ‐acetylcysteine were only detected in BSE group mice by ultra‐performance liquid chromatography‐MS analysis. In addition, BSE intervention evidently increased the abundance of Alistipeds , Coriobacteriaceae UCG‐002 and Bifidobacterium and decreased the abundance of Escheichia‐Shinella , Lachnospiraceae others , Parabacteroides , Ruminococcaceae others and Turicibacter , which possibly promoted carbohydrate metabolism and short‐chain fatty acid production. CONCLUSION The present study aimed to elucidate the effect of BSE on colitis and found that BSE, as a novel food ingredient, has great potential for the improvement of colitis. © 2022 Society of Chemical Industry.

Wu, Jiaying↗
DOE OSTI2022

Biomass formation and sugar release efficiency of Populus modified by altered expression of a NAC transcription factor

Woody biomass is an important feedstock for biofuel production. Manipulation of wood properties that enable efficient conversion of biomass to biofuel reduces cost of biofuel production. Wood cell wall composition is regulated at several levels that involve expression of transcription factors such as wood-/secondary cell wall-associated NAC domains (WND or SND). In Arabidopsis thaliana, SND1 regulates cell wall composition through activation of its down-stream targets such as MYBs. The functional aspects of SND1 homologs in the woody Populus have been studied through transgenic manipulation. In this study, we investigated the role of PdWND1B, Populus SND1 sequence ortholog, in wood formation using transgenic manipulation through over-expression or silencing under the control of a vascular-specific 4-coumarate-CoA ligase (4CL) promoter. As compared with control plants, PdWND1B-RNAi plants were shorter in height, with significantly reduced stem diameter and dry biomass, whereas there were no significant differences in growth and productivity of PdWND1B over-expression plants. Conversely, PdWND1B over-expression lines showed a significant reduction in cellulose and increase in lignin content, whereas there was no significant impact on lignin content of downregulated lines. Stem carbohydrate composition analysis revealed a decrease in glucose, mannose, arabinose, and galactose, but an increase in xylose in the over-expression lines. Transcriptome analysis revealed upregulation of several downstream transcription factors and secondary cell wall related structural genes in the PdWND1B over-expression lines, partly explaining the observed phenotypic changes in cell wall chemistry. Relative to the control, glucose release efficiency and ethanol production from stem biomass was significantly reduced in over-expression lines. Our results show that PdWND1B is an important factor determining biomass productivity, cell wall chemistry and its conversion to biofuels in Populus.

09 BIOMASS FUELS↗
DOE OSTI2021

Structural elucidation and immuno-stimulatory activity of a novel polysaccharide containing glucuronic acid from the fungus Echinodontium tinctorium

An immuno-stimulatory polysaccharide (EtISPFa) was purified from water extract of the fungus Echinodontium tinctorium. EtISPFa has an estimated weight average molecular weight (M w ) of 1354 kDa and is composed of glucose (66.2 %), glucuronic acid (10.1 %), mannose (6.7 %), galactose (6.4 %), xylose (5.6 %), rhamnose (3.1 %), fucose (1.8 %), and arabinose (0.2 %). It has multiple glycosidic linkages, with 3-Glcp (19.8 %), 4-GlcpA (10.8 %), 6-Glcp (10.7 %), and 3,6-Glcp (8.7 %) being the most prominent. NMR analysis showed that EtISPFa has a backbone containing mostly of 3-substituted β-glucopyranose with 4-substituted glucopyranosyluronic acid. Short side chains consisting of an average of two β-glycopyranose residues, connected through 1→6 linkages, are attached to the 6-position of about every 4th or 5th backbone glucose residue. EtISPFa is a novel glucuronic acid-containing β-glucan capable of significantly inducing the production of cytokines IL-17, IL-16, MIP-2, G-CSF,GM-CSF, LIF, MIP-1α, MIP-1β, and RANTES in vitro. EtISPFa should be further explored for its immuno-stimulatory activity in vivo.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Hempseed cell wall polysaccharides are dominated by linear xylans and cellulose: Comprehensive structural profiling of ten cultivars of industrial hemp, Cannabis sativa L .

Hempseed is a rich source of dietary fiber; however, there has been limited research on the variability of carbohydrate composition in hempseed cell walls. The primary aim of this study was to conduct a comprehensive chemical and structural analysis of the cell wall polysaccharides in ten hempseed cultivars. Water-soluble polysaccharides (WSP) and water-insoluble residues (WIR) were isolated and subsequently analyzed for their monosaccharide composition using HPAEC-PAD, glycosyl linkage analysis using GC–MS, and structural characterization via NMR spectroscopy. All hempseed cultivars contained a high proportion of insoluble fibers and smaller amounts of soluble polysaccharides. Glucose and xylose were the most abundant components of the WIR fractions, while the WSP fractions contained abundant amounts of galactose, galacturonic acid, arabinose, rhamnose, and mannose. The results of linkage and spectroscopic analysis were consistent with the compositional analysis, identifying cellulose and acetylated linear xylans as primary components of WIR, and arabinogalactans, rhamnogalacturonans, heteromannans, xyloglucans, and arabinan as predominant in WSP. Altogether, the study revealed a comparable cell wall structure among the analyzed hemp seed varieties. The high fiber content of whole hempseed-based ingredients presents significant potential for food manufacturers seeking to develop products with enhanced dietary fiber content, offering both functional and nutritional benefits for consumers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

The pectin puzzle: Decoding the fine structure of rhamnogalacturonan-I (RG-I) in Arabidopsis thaliana uncovers new pectin features

Pectin is generally divided into four distinct structural categories, namely homogalacturonan, xylogalacturonan, rhamnogalacturonan I (RG-I) and rhamnogalacturonan II. While much of the structural diversity of homogalacturonan, xylogalacturonan and rhamnogalacturonan II has been elucidated, the structural features of RG-I are less well understood. In this work, we employed multiple complementary analytical techniques to present a detailed structural analysis of RG-I in the model species Arabidopsis thaliana . Starting with highly purified RG-I from different Arabidopsis tissues, we employed comparative linkage and nuclear magnetic resonance analysis along with mass spectrometry analysis of enzymatically digested RG-I oligosaccharides. Besides the presence of the canonical α-1,5-arabinan, β-1,4-galactan, β-1,6-galactan and arabinogalactan RG-I side chains of varying lengths, we show that a large portion of the β-1,6-galactan is terminated by either 4-O-methyl β-glucuronic acid (GlcA) residues or, to a smaller degree, β-GlcA that lacks the Me-ether group. Importantly, O-acetylation of RG-I GalA residues is a minor modification while 10 % of the backbone Rha residues are 3-O-acetylated, and most of the acetylated Rha is additionally branched with β-galactose substituents. Taken together, the combined results of these different analytical techniques present the most comprehensive structural overview of Arabidopsis thaliana RG-I to date.

25 ENERGY STORAGE↗
DOE OSTI2020

An acidic polysaccharide (AGC3) isolated from North American ginseng ( Panax quinquefolius ) suspension culture as a potential immunomodulatory nutraceutical

Polysaccharides isolated from Panax quinquefolius roots are widely used as nutraceuticals due to their immunomodulatory properties. Despite their popularity, several challenges exist in isolating ginseng root polysaccharides such as batch-to-batch structural inconsistencies and bacterial endotoxin contamination. A plant tissue culture-based platform offers a potential solution to isolate natural polysaccharide fractions with consistent chemical characteristics and reduced endotoxin content. In this study, an acidic polysaccharide fraction (AGC3) with immunomodulatory properties was isolated from Panax quinquefolius suspension cultures. The heterogeneous fraction (molecular weight: 4.81 and 32.14 kDa), purified by anion exchange chromatography, was predominantly composed of galactose (>60%) along with the presence of rhamnose, arabinose, glucose, glucuronic acid and galacturonic acid. The major glycosidic linkages were found to be t-Galp (47.7%), 4-Galp (15.6%), 2,4-Rhap (8.1%), 6-Galp (8.1%) and 4-GalAp (6.8%). Structural analyses indicated the presence of a pectic rhamnogalacturonan I polysaccharide in AGC3. AGC3 significantly (p < 0.05) stimulated RAW 264.7 murine macrophage cells and primary murine splenocytes by enhancing the production of several immunomodulatory mediators such as IL-6, TNF-α, GM-CSF and MCP-1. The results also indicated the putative roles of NF-κB (p65/RelA) and MAPK (p38) signaling pathways in the immunostimulatory response. Additionally, AGC3 induced murine splenocyte proliferation, another major indicator of immunostimulation. Overall, AGC3 has the potential to be used as an immunostimulatory nutraceutical.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Characterization of two types of polysaccharides from Eremurus hissaricus roots growing in Tajikistan

Eremurus Hissaricus (E.hissaricus) is a native plant of Tajikistan that has been used in traditional medicine and whose raw components have been identified as having potential for the domestic economy. We have found that carbohydrates comprise 80–85% of the root biomass, from which we have characterized two types of polysaccharides: A water-soluble polysaccharide (WSP) and an acid-soluble polysaccharide (ASP), with 23% and 8% yields, respectively. The results from FTIR, HPAEC-PAD, HPSEC, methylation linkage analysis and NMR spectroscopy ( 1 H, 13 C, zTOCSY, HSQC, HSQCTOXY and HMBC) demonstrated that WSP has a backbone of (1 → 4)-linked β-d-mannopyranosyl and β-d-glucopyranosyl residues in a 1.5:1 M ratio, having acetylated groups, with weight-average molecular weights of 495 kDa. Analyses of ASP indicate that it is comprised of d-galactose, d-mannose, and d-glucose in a molar ratio of 1.8: 1.6: 1.0, which is consistent with a branched galactoglucomannan polysaccharide with (1 → 4)-linked β-pyranose configurations, and weight-average molecular weight of 179 kDa. The FTIR absorption bands in the region of 875–890 cm 1 and NMR spectroscopy data confirm the presence of β-pyranose configuration of main polysaccharide backbone in the both ASP and WSP samples. These polysaccharides are an available raw material with the potential for use in a number of industrial applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structure and composition of blueberry fiber pectin and xyloglucan that bind anthocyanins during fruit puree processing

Blueberry is a well-known source of antioxidants and prebiotic dietary fiber yet the fate of these health-beneficial ingredients during fruit processing it is not understood. Detailed composition and structure of blueberry pomace (PF) remaining after commercial blueberry fruit puree processing, as well as the water soluble and water insoluble fractions, were determined. The PF was comprised of dietary fiber (60.8%) that was largely insoluble (46.2% vs. 14.6% soluble dietary fiber) pectin, xyloglucan, arabinoxylan and mannan polysaccharides, fructose (11.22%) and glucose (10.37%) free sugars, protein (9%), fat (5%), and anthocyanins (6444.5 μg/g). The most abundant anthocyanins detected in PF were malvidin 3-arabinoside and peonidin 3-glucoside and the pectin was determined to be methyl-esterified and acetylated rhamnogalacturonan I with a 4,5-unsaturated function at the non-reducing end based on oligosaccharide structural analysis. Blueberry PF xyloglucan oligosaccharide side chain structures included xylose, arabinose and galactose consistent with arabino-xyloglucan structure. Additionally, microwave-assisted extraction of blueberry PF (pH 2 for 10 min at 80 °C) was performed to more clearly elucidate pectin structure, and revealed a pectin polymer with high molar mass (1072 kDa) and viscosity that was dependent upon the molar mass. A random coil shape was confirmed for both the blueberry PF and microwave extracted blueberry pectin, which agreed with previous reports that flexible blueberry pectin contributes to strong anthocyanin binding. In summary, the blueberry pomace anthocyanin content was slightly higher than that reported for fresh whole blueberry fruit, which suggests that most blueberry anthocyanins adhered to the pomace fiber, and most likely the insoluble pectin, during and after processing.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Structural characterization of strawberry pomace

Strawberries are a nutrient dense food rich in vitamins, minerals, non-nutrient antioxidant phenolics, and fibers. Strawberry fiber bioactive structures are not well characterized and limited information is available about the interaction between strawberry fiber and phenolics. Therefore, we analyzed commercial strawberry pomace in order to provide a detailed carbohydrate structural characterization, and to associate structures with functions. The pomace fraction, which remained after strawberry commercial juice extraction, contained mostly insoluble (49.1 % vs. 5.6 % soluble dietary fiber) dietary fiber, with pectin, xyloglucan, xylan, β-glucan and glucomannan polysaccharides; glucose, fructose, xylose, arabinose, galactose, fucose and galacturonic acid free carbohydrates; protein (15.6 %), fat (8.34 %), and pelargonidin 3-glucoside (562 μg/g). Oligosaccharides from fucogalacto-xyloglucan, methyl-esterified rhamnogalacturonan I with branched arabinogalacto-side chains, rhamnogalacturonan II, homogalacturonan and β-glucan were detected by MALDI-TOF MS, NMR and glycosyl-linkage analysis. Previous reports suggest that these oligosaccharide and polysaccharide structures have prebiotic, bacterial pathogen anti-adhesion, and cholesterol-lowering activity, while anthocyanins are well-known antioxidants. A strawberry pomace microwave acid-extracted (10 min, 80 °C) fraction had high molar mass (2376 kDa) and viscosity (3.75 dL/g), with an extended rod shape. A random coil shape, that was reported previously to bind to phenolic compounds, was observed for other strawberry microwave-extracted fractions. These strawberry fiber structural details suggest that they can thicken foods, while the polysaccharide and polyphenol interaction indicates great potential as a multiple-function bioactive food ingredient important for gut and metabolic health.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Borate-assisted alkaline extraction of hemicellulose from switchgrass with enhanced structural stability and purity

Valorization of non-cellulosic polysaccharides is crucial for enhancing the economic competitiveness of biorefinery processes. In this study, a mixture of boric acid and sodium hydroxide was employed to efficiently extract hemicellulose from holocellulose switchgrass. Borate-assisted alkaline extraction resulted in a higher xylan content (59.5 %) compared to conventional alkaline extraction. Here, the hemicellulose fractions derived from the borate-alkaline treatment exhibited a higher molecular weight (M w = 51.2 kDa) and a relatively lower degree of polydispersity (1.28), indicating improved structural stability. The presence of borate had a protective effect against chain scission, preserving glucuronic acid residues and increasing galactose content. Additionally, borate improved hemicellulose purity, with up to 74.1 % of the extracted hemicellulose being suitable for further enzymatic applications. Extended extraction time further enhanced hemicellulose recovery, reaching 97.9 % under NaOH/boric acid conditions while maintaining structural integrity, as confirmed by SEM, FTIR and 2D HSQC NMR analyses. These findings provide insights into the role of borate in optimizing hemicellulose extraction and improving its potential for bioconversion processes.

Borate alkaline↗
DOE OSTI2022

Bioinspired oxidation of benzyl alcohol: The role of environment and nuclearity of the catalyst evaluated by multivariate analysis

Inspired by copper-containing enzymes such as galactose oxidase and catechol oxidase, in which distinct coordination environments and nuclearities lead to specific catalytic activities, we summarize here the catalytic properties of dinuclear and mononuclear copper species towards benzyl alcohol oxidation using a multivariate statistical approach. Here, the new dinuclear [Cu 2 (μ-L 1 )(μ-pz)] 2+ (1) is compared against the mononuclear [CuL 2 Cl] (2), where (L 1 ) - and (L 2 ) - are the respective deprotonated forms of 2,6-bis((bis(pyridin-2-ylmethyl)amino)methyl)-4-methylphenol, and 3-((bis(pyridin-2-ylmethyl)amino)methyl)-2-hydroxy-5-methylbenzaldehyde and (pz) - is a pyrazolato bridge. Copper(II) perchlorate (CP) is used as control. The catalytic oxidation of benzyl alcohol is pursued, aiming to assess the role of the ligand environment and nuclearity. The multivariate statistical approach allows for the search of optimal catalytic conditions, considering variables such as catalyst load, hydrogen peroxide load, and time. Species 1, 2 and CP promoted selective production of benzaldehyde at different yields, with only negligible amounts of benzoic acid. Under normalized conditions, 2 showed superior catalytic activity. This species is 3.5-fold more active than the monometallic control CP, and points out to the need for an efficient ligand framework. Species 2 is 6-fold more active than the dinuclear 1, and indicates the favored nuclearity for the conversion of alcohols into aldehydes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Verazine biosynthesis from simple sugars in engineered Saccharomyces cerevisiae

Steroidal alkaloids are FDA-approved drugs (e.g., Zytiga) and promising drug candidates/leads (e.g., cyclopamine); yet many of the ≥697 known steroidal alkaloid natural products remain underutilized as drugs because it can be challenging to scale their biosynthesis in their producing organisms. Cyclopamine is a steroidal alkaloid produced by corn lily (Veratrum spp.) plants, and it is an inhibitor of the Hedgehog (Hh) signaling pathway. Therefore, cyclopamine is an important drug candidate/lead to treat human diseases that are associated with dysregulated Hh signaling, such as basal cell carcinoma and acute myeloid leukemia. Cyclopamine and its semi-synthetic derivatives have been studied in (pre)clinical trials as Hh inhibitor-based drugs. However, challenges in scaling the production of cyclopamine have slowed efforts to improve its efficacy and safety profile through (bio)synthetic derivatization, often limiting drug development to synthetic analogs of cyclopamine such as the FDA-approved drugs Odomzo, Daurismo, and Erivedge. If a platform for the scalable and sustainable production of cyclopamine were established, then its (bio)synthetic derivatization, clinical development, and, ultimately, widespread distribution could be accelerated. Ongoing efforts to achieve this goal include the biosynthesis of cyclopamine in Veratrum plant cell culture and the semi-/total chemical synthesis of cyclopamine. Herein, this work advances efforts towards a promising future approach: the biosynthesis of cyclopamine in engineered microorganisms. We completed the heterologous microbial production of verazine (biosynthetic precursor to cyclopamine) from simple sugars (i.e., glucose and galactose) in engineered Saccharomyces cerevisiae (S. cerevisiae) through the inducible upregulation of the native yeast mevalonate and lanosterol biosynthetic pathways, diversion of biosynthetic flux from ergosterol (i.e., native sterol in S. cerevisiae) to cholesterol (i.e., biosynthetic precursor to verazine), and expression of a refactored five-step verazine biosynthetic pathway. The engineered S. cerevisiae strain that produced verazine contains eight heterologous enzymes sourced from seven different species. Importantly, S. cerevisiae-produced verazine was indistinguishable via liquid chromatography-mass spectrometry from both a commercial standard (Veratrum spp. plant-produced) and Nicotiana benthamiana-produced verazine. To the best of our knowledge, this is the first report describing the heterologous production of a steroidal alkaloid in an engineered yeast. Verazine production was ultimately increased through design-build-test-learn cycles to a final titer of 83 ± 3 μg/L (4.1 ± 0.1 μg/g DCW). Finally, this research lays the groundwork for future microbial biosynthesis of cyclopamine, (bio)synthetic derivatives of cyclopamine, and other steroidal alkaloid natural products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Carbohydrate-Containing Conjugated Polymers: Solvent-Resistant Materials for Greener Organic Electronics

Organic semiconducting polymers are exciting materials for electronic applications because of their good mechanical and optoelectronic properties. A major advantage of organic semiconductors is their solution processability. This allows access to a variety of simple and cost-effective device fabrication methods compared to the expensive, high-temperature processing methods required for silicon-based electronics. However, these materials often have low solubility, which limits their processing to toxic halogenated solvents. Also, their limited solubility often leads to interfacial mixing during device fabrication. Herein, this work explores the incorporation of environmentally friendly carbohydrate side chains in conjugated polymers to enhance processability in eco-friendly solvents. Moreover, a mild postprocessing treatment was designed to enable solvent resistance. Isoindigo-based polymers with varied ratios of acetyl-protected galactose side chains were synthesized to improve solubility in o-anisole in the protected state, while inducing solvent resistance through intramolecular hydrogen bonding in the deprotected state. Solvent resistance was confirmed both visually upon submersion in various solvents and using UV–visible spectroscopy. Importantly, the mild basic treatment to achieve solvent resistance has no negative impact on the electronic performance of these materials in organic field-effect transistors, even after subsequent submersion in various solvents, making them a valuable platform for the production of green processable multilayer electronics.

36 MATERIALS SCIENCE↗