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At least 127 records · Page 7

Data for Cloning and Characterization of a Panel of Mitochondrial Targeting Sequences for Compartmentalization Engineering in Saccharomyces cerevisiae

Mitochondrion is generally considered as the most promising subcellular organelle for compartmentalization engineering. Much progress has been made in reconstituting whole metabolic pathways in the mitochondria of yeast to harness the precursor pools (i.e., pyruvate and acetyl-CoA), bypass competing pathways, and minimize transportation limitations. However, only a few mitochondrial targeting sequences (MTSs) have been characterized (i.e., MTS of COX4), limiting the application of compartmentalization engineering for multigene biosynthetic pathways in the mitochondria of yeast. In the present study, based on the mitochondrial proteome, a total of 20 MTSs were cloned and the efficiency of these MTSs in targeting heterologous proteins, including the Escherichia coli FabI and enhanced green fluorescence protein (EGFP) into the mitochondria was evaluated by growth complementation and confocal microscopy. After systematic characterization, six of the well-performed MTSs were chosen for the colocalization of complete biosynthetic pathways into the mitochondria. As proof of concept, the full α-santalene biosynthetic pathway consisting of 10 expression cassettes capable of converting acetyl-coA to α-santalene was compartmentalized into the mitochondria, leading to a 3.7-fold improvement in the production of α-santalene. The newly characterized MTSs should contribute to the expanded metabolic engineering and synthetic biology toolbox for yeast mitochondrial compartmentalization engineering.

Conversion↗

Data for Glucose Feeds the Tricarboxylic Acid Cycle via Excreted Ethanol in Fermenting Yeast

Ethanol and lactate are typical waste products of glucose fermentation. In mammals, glucose is catabolized by glycolysis into circulating lactate, which is broadly used throughout the body as a carbohydrate fuel. Individual cells can both uptake and excrete lactate, uncoupling glycolysis from glucose oxidation. Here we show that similar uncoupling occurs in budding yeast batch cultures of Saccharomyces cerevisiae and Issatchenkia orientalis . Even in fermenting S. cerevisiae that is net releasing ethanol, media 13C-ethanol rapidly enters and is oxidized to acetaldehyde and acetyl-CoA. This is evident in exogenous ethanol being a major source of both cytosolic and mitochondrial acetyl units. 2H-tracing reveals that ethanol is also a major source of both NADH and NADPH high-energy electrons, and this role is augmented under oxidative stress conditions. Thus, uncoupling of glycolysis from the oxidation of glucose-derived carbon via rapidly reversible reactions is a conserved feature of eukaryotic metabolism.

Conversion↗

Data for Engineering and Evolution of Yarrowia lipolytica for Producing Lipids from Lignocellulosic Hydrolysates

Yarrowia lipolytica , an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates.

Hydrolysate↗

Data for Complete and Efficient Conversion of Plant Cell Wall Hemicellulose into High-Value Bioproducts by Engineered Yeast

Plant cell wall hydrolysates contain not only sugars but also substantial amounts of acetate, a fermentation inhibitor that hinders bioconversion of lignocellulose. Despite the toxic and non-consumable nature of acetate during glucose metabolism, we demonstrate that acetate can be rapidly co-consumed with xylose by engineered Saccharomyces cerevisiae . The co-consumption leads to a metabolic re-configuration that boosts the synthesis of acetyl-CoA derived bioproducts, including triacetic acid lactone (TAL) and vitamin A, in engineered strains. Notably, by co-feeding xylose and acetate, an engineered strain produces 23.91 g/L TAL with a productivity of 0.29 g/L/h in bioreactor fermentation. This strain also completely converts a hemicellulose hydrolysate of switchgrass into 3.55 g/L TAL. These findings establish a versatile strategy that not only transforms an inhibitor into a valuable substrate but also expands the capacity of acetyl-CoA supply in S. cerevisiae for efficient bioconversion of cellulosic biomass.

Conversion↗

Data for "Decompartmentalization of the yeast mitochondrial metabolism to improve chemical production in Issatchenkia orientalis "

Microbial production of chemicals may suffer from inadequate cofactor provision, a challenge further exacerbated in yeasts due to compartmentalized cofactor metabolism. Here, we perform cofactor engineering through the decompartmentalization of mitochondrial metabolism to improve succinic acid (SA) production in Issatchenkia orientalis . We localize the reducing equivalents of mitochondrial NADH to the cytosol through cytosolic expression of its pyruvate dehydrogenase (PDH) complex and couple a reductive tricarboxylic acid pathway with a glyoxylate shunt, partially bypassing an NADH-dependent malate dehydrogenase to conserve NADH. Cytosolic SA production reaches a titer of 104 g/L and a yield of 0.85 g/g glucose, surpassing the yield of 0.66 g/g glucose constrained by cytosolic NADH availability. Additionally, expressing cytosolic PDH, we expand our I. orientalis platform to enhance acetyl-CoA-derived citramalic acid and triacetic acid lactone production by 1.22- and 4.35-fold, respectively. Our work establishes I. orientalis as a versatile platform to produce markedly reduced and acetyl-CoA-derived chemicals.

bioproducts↗

Evaluating the Persistence of Soil Carbon After a 30-Year Nitrogen Fertilization Experiment at the Fernow Experimental Forest, WV

We sampled soils in a previously nitrogen (N)-fertilized watershed (Watershed 3), which received 35 kg N per hectare annually for 30 years, and a reference watershed (Watershed 7), 4 years after the end of a 30-year N fertilization experiment at the Fernow Experimental Forest in West Virginia. As N deposition has reduced soil pH and plant carbon (C) investments into the rhizosphere, we compared the extent to which removing these potential limitations to microbial decomposition by increasing soil pH, adding artificial root exudates, or elevating soil temperature would increase microbial decomposition (and soil C losses) in soils that have and have not received excess N inputs. As such, we incubated soils with and without the addition of artificial root exudates and dolomitic lime (2 amendment treatments and 1 untreated control), at three different temperatures (15, 20, and 25 C) to simulate warming. We measured soil respiration, enzyme activity (acid phosphatase, N -acetyl-β-glucosaminidase, beta-glucosidase, phenol oxidase, and peroxidase), and microbial biomass carbon in a 15-week microcosm experiment. All of the datasheets include the metadata for all of the treatment combinations from this incubation experiment (ID, incubation temperature, treatment, watershed).The "Soil_Chemistry_Data.csv" data includes (1) the ratio of dry soil weight (g) by wet (or fresh) soil weight (g) after 72 hours at 65 C, (2) the wet (or fresh) weight of the soil samples used to determine soil pH, and (3) soil pH, which was measured in CaCl2. Detailed methods for the measurements of soil pH are described in the datasheet itself.The "Respiration_Data.csv" data includes (1) respiration day for the measurement, and (2) cumulative CO2 respiration (mmol) measured in the microcosms on specific days.The "Microbial_Biomass_Data.csv" data includes (1) mmol microbial biomass carbon per g (dry weight) of soil in each microcosm.The "Enzyme_Data.csv" data includes (1) all extracellular soil enzyme activity (N-acetyl-glucosaminidase (NAG), acid phosphatase (AP), β-glucosidase (BG), phenol oxidase and peroxidase) that we measured in our microcosms.

54 ENVIRONMENTAL SCIENCES↗

Using 14 C-acetate Pulse-chase Labeling to Study Fatty Acid and Glycerolipid Metabolism in Plant Leaves

Lipids metabolism is comprised of networks of reactions occurred in different subcellular compartments. Isotopic labeling is a good way to track the transformations and movements of metabolites without perturbing overall cellular metabolism. Fatty acids, the building blocks of membrane lipids and storage triacylglycerols, are synthesized in plastids. The immediate precursor for fatty acid synthesis is acetyl-CoA. Exogenous acetate is rapidly incorporated into fatty acids in leaves and isolated plastids because it can diffuse freely through cellular membranes, enter the plastid where it is rapidly metabolized to acetyl-CoA. Therefore, isotope-labeled acetate is often used as a tracer for the investigation of fatty acid synthesis and complex lipid metabolism in plants and other organisms. The basic principle of isotope labeling and its recent technical advances have been reviewed (Allen et al., 2015). The present protocol describes the use of 14C-labeled acetate to determine rates of fatty acid synthesis and degradation and to track the metabolism of glycerolipids in leaves. This method, which is often referred to as acetate pulse-chase labeling, has been widely used to probe various aspects of lipid metabolism (Allen et al., 2015), including the role of autophagy in membrane lipid turnover (Fan et al., 2019) and the interplay between lipid and starch metabolism pathways (Yu et al., 2018).

14C-acetate pulse-chase↗

Lignin molecular weights of Populus trichocarpa residues after CELF pretreatment

Here we present a dataset of molecular weights of lignin from a woody energy crop (Populus trichocarpa) residues after a series of co-solvent enhanced lignocellulosic fractionation (CELF) pretreatment. The natural poplar variant GW-9947 from the Center for Bioenergy Innovation (CBI) was used. The poplar was knife milled and passed through a 1 mm sieve and CELF pretreatment was performed in a Parr autoclave reactor with 7.5 wt % solids loading, 0.5 wt% H2SO4 as catalyst at 150°C with various time. Tetrahydrofuran was added in a 1:1 mass ratio with water as the pretreatment solvent. Lignin was isolated from the pretreated samples after ball-milling in a porcelain jar with ceramic balls via Retsch PM 200 at 580 rpm for 2.5 h followed by enzymatic hydrolysis in acetate buffer (pH 4.8, 50 °C) for 48 h. The solid residue was isolated by centrifugation and hydrolyzed again with freshly added buffer and enzymes for another 48 h. After filtration, the solid residue was extracted twice with 96% (v/v) 1,4-dioxane/water mixture at room temperature overnight. The extracts were combined, rotary evaporated, and freeze-dried to recover lignin. The lignin samples were then derivatized in an acetic anhydride/pyridine (1:1, v/v) mixture and stirred at room temperature for 24 h. Ethanol was added to the reaction mixture, left for 30 min and then removed with a rotary evaporator. The addition and removal of ethanol was repeated at least 3 times until all traces of acetic acid were removed. Acetylated lignin samples were then dissolved in tetrahydrofuran (THF) at a concentration of 1.0 mg/mL. The molecular weight of acetylated lignin was measured by a gel permeation chromatography (GPC) on a PSS-Polymer Standards Service (Warwick, RI, USA) GPC SECurity 1200 system featuring Agilent HPLC 1200 components equipped with four Waters Styragel columns (HR1, HR2, HR4 and HR6) and an UV detector (270 nm). Tetrahydrofuran was used as the mobile phase and flow rate was 0.3 mL/min. The Polymer Standards Service WinGPC Unity software (Build 6807) was used for data processing for all the samples. The data provides information about the effects of CELF pretreatment time at 150 ºC on lignin molecular weights.

09 BIOMASS FUELS↗

Potential Activities and Long Lifetimes of Organic Carbon-Degrading Extracellular Enzymes in Deep Subsurface Sediments of the Baltic Sea

Heterotrophic microorganisms in marine sediments produce extracellular enzymes to hydrolyze organic macromolecules, so their products can be transported inside the cell and used for energy and growth. Therefore, extracellular enzymes may mediate the fate of organic carbon in sediments. The Baltic Sea Basin is a primarily depositional environment with high potential for organic matter preservation. The potential activities of multiple organic carbon-degrading enzymes were measured in samples obtained by the International Ocean Discovery Program Expedition 347 from the Little Belt Strait, Denmark, core M0059C. Potential maximum hydrolysis rates (V max ) were measured at depths down to 77.9mbsf for the following enzymes: alkaline phosphatase, β- d -xylosidase, β- d -cellobiohydrolase, N-acetyl-β- d -glucosaminidase, β-glucosidase, α-glucosidase, leucyl aminopeptidase, arginyl aminopeptidase, prolyl aminopeptidase, gingipain, and clostripain. Extracellular peptidase activities were detectable at depths shallower than 54.95mbsf, and alkaline phosphatase activity was detectable throughout the core, albeit against a relatively high activity in autoclaved sediments. β-glucosidase activities were detected above 30mbsf; however, activities of other glycosyl hydrolases (β-xylosidase, β-cellobiohydrolase, N-acetyl-β-glucosaminidase, and α-glucosidase) were generally indistinguishable from zero at all depths. These extracellular enzymes appear to be extremely stable: Among all enzymes, a median of 51.3% of enzyme activity was retained after autoclaving for an hour. We show that enzyme turnover times scale with the inverse of community metabolic rates, such that enzyme lifetimes in subsurface sediments, in which metabolic rates are very slow, are likely to be extraordinarily long. A back-of-the-envelope calculation suggests enzyme lifetimes are, at minimum, on the order of 230days, and may be substantially longer. These results lend empirical support to the hypothesis that a population of subsurface microbes persist by using extracellular enzymes to slowly metabolize old, highly degraded organic carbon.

54 ENVIRONMENTAL SCIENCES↗

Dynamic acylome reveals metabolite driven modifications in Syntrophomonas wolfei

Syntrophomonas wolfei is an anaerobic syntrophic microbe that degrades short-chain fatty acids to acetate, hydrogen, and/or formate. This thermodynamically unfavorable process proceeds through a series of reactive acyl-Coenzyme A species (RACS). In other prokaryotic and eukaryotic systems, the production of intrinsically reactive metabolites correlates with acyl-lysine modifications, which have been shown to play a significant role in metabolic processes. Analogous studies with syntrophic bacteria, however, are relatively unexplored and we hypothesized that highly abundant acylations could exist in S. wolfei proteins, corresponding to the RACS derived from degrading fatty acids. Here, by mass spectrometry-based proteomics (LC–MS/MS), we characterize and compare acylome profiles of two S. wolfei subspecies grown on different carbon substrates. Because modified S. wolfei proteins are sufficiently abundant to analyze post-translational modifications (PTMs) without antibody enrichment, we could identify types of acylations comprehensively, observing six types (acetyl-, butyryl-, 3- hydroxybutyryl-, crotonyl-, valeryl-, and hexanyl-lysine), two of which have not been reported in any system previously. All of the acyl-PTMs identified correspond directly to RACS in fatty acid degradation pathways. A total of 369 sites of modification were identified on 237 proteins. Structural studies and in vitro acylation assays of a heavily modified enzyme, acetyl-CoA transferase, provided insight on the potential impact of these acyl-protein modifications. The extensive changes in acylation-type, abundance, and modification sites with carbon substrate suggest that protein acylation by RACS may be an important regulator of syntrophy.

Fu, Janine Y.↗

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↗

Reconfiguring Plant Metabolism for Biodegradable Plastic Production

For decades, plants have been the subject of genetic engineering to synthesize novel, value-added compounds. Polyhydroxyalkanoates (PHAs), a large class of biodegradable biopolymers naturally synthesized in eubacteria, are among the novel products that have been introduced to make use of plant acetyl-CoA metabolic pathways. It was hoped that renewable PHA production would help address environmental issues associated with the accumulation of nondegradable plastic wastes. However, after three decades of effort synthesizing PHAs, and in particular the simplest form polyhydroxybutyrate (PHB), and seeking to improve their production in plants, it has proven very difficult to reach a commercially profitable rate in a normally growing plant. This seems to be due to the growth defects associated with PHA production and accumulation in plant cells. Here, we review major breakthroughs that have been made in plant-based PHA synthesis using traditional genetic engineering approaches and discuss challenges that have been encountered. Then, from the point of view of plant synthetic biology, we provide perspectives on reprograming plant acetyl-CoA pathways for PHA production, with the goal of maximizing PHA yield while minimizing growth inhibition. Specifically, we suggest genetic elements that can be considered in genetic circuit design, approaches for nuclear genome and plastome modification, and the use of multiomics and mathematical modeling in understanding and restructuring plant metabolic pathways.

59 BASIC BIOLOGICAL SCIENCES↗

Structure and flexibility of the yeast NuA4 histone acetyltransferase complex

The NuA4 protein complex acetylates histones H4 and H2A to activate both transcription and DNA repair. We report the 3.1-Å resolution cryo-electron microscopy structure of the central hub of NuA4, which flexibly tethers the histone acetyltransferase (HAT) and Trimer Independent of NuA4 involved in Transcription Interactions with Nucleosomes (TINTIN) modules. The hub contains the large Tra1 subunit and a core that includes Swc4, Arp4, Act1, Eaf1, and the C-terminal region of Epl1. Eaf1 stands out as the primary scaffolding factor that interacts with the Tra1, Swc4, and Epl1 subunits and contributes the conserved HSA helix to the Arp module. Using nucleosome-binding assays, we find that the HAT module, which is anchored to the core through Epl1, recognizes H3K4me3 nucleosomes with hyperacetylated H3 tails, while the TINTIN module, anchored to the core via Eaf1, recognizes nucleosomes that have hyperacetylated H2A and H4 tails. Together with the known interaction of Tra1 with site-specific transcription factors, our data suggest a model in which Tra1 recruits NuA4 to specific genomic sites then allowing the flexible HAT and TINTIN modules to select nearby nucleosomes for acetylation.

59 BASIC BIOLOGICAL SCIENCES↗

Methods for detecting and determining levels of monolignol ester conjugates incorporated into lignin and compounds relating thereto

Methods of detecting and, optionally, determining a level of incorporation of monolignol ester conjugates into lignin. The methods include derivatizing lignin to acylate at least a portion of free phenolic and aliphatic hydroxyls and to halogenate at least a portion of benzylic alcohols present in the lignin to yield derivatized lignin, treating the derivatized lignin with a reducing agent to cleave at least a portion of the derivatized lignin to yield lignin cleavage products, acetylating at least a portion of free hydroxyl groups in the lignin cleavage products with a labeled acetylation agent to yield labeled lignin fragments, and detecting the labeled lignin fragments.

09 BIOMASS FUELS↗

Recombinant microorganisms and uses therefor

Microorganisms are genetically engineered to produce 3-hydroxypropionate (3-HP). The microorganisms are carboxydotrophic acetogens. The microorganisms produce acetyl-coA using the Wood-Ljungdahl pathway for fixing CO/CO 2 . A β-alanine pyruvate aminotransferase from a microorganism that contains such an enzyme is introduced. Additionally, an acetyl-coA carboxylase may also be introduced. The production of 3-HP can be improved. This can be effected by improved promoters or higher copy number or enzymes that are catalytically more efficient.

Liew, Fungmin↗

A Thermosensitive, Phase-Variable Epigenetic Switch: pap Revisited

It has been more than a decade since the last comprehensive review of the phase-variable uropathogen-associated pyelonephritis-associated pilus (pap) genetic switch. Since then, important data have come to light, including additional factors that regulate pap expression, better characterization of H-NS regulation, the structure of the Lrp octamer in complex with pap regulatory DNA, the temperature-insensitive phenotype of a mutant lacking the acetyltransferase RimJ, evidence that key components of the regulatory machinery are acetylated, and new insights into the role of DNA binding by key regulators in shaping both the physical structure and regulatory state of the papI and papBA promoters. This review revisits pap, integrating these newer observations with older ones to produce a new model for the concerted behavior of this virulence-regulatory region.

gene silencing↗

Acylation as a successful derivatization strategy for the analysis of pinacolyl alcohol in a glycerol-rich matrix by GC-MS: application during an OPCW Proficiency Test

A derivatization protocol based on the acylation of pinacolyl alcohol (PA), an important marker for the nerve agent soman, is presented. The procedure provides a convenient means of detecting, by gas chromatography-mass spectrometry (GC-MS), PA when present at a low concentration in a complex glycerol/alcohol-rich matrix. While there are only two reports describing the specific analysis of PA in matrices at low concentrations, the protocol described herein represents the first of its kind in the analysis of PA in a highly reactive matrix. Two alternative paths for the protocol’s execution are presented. The first involves the direct derivatization of the PA with either acetyl or benzoyl chloride; both reactions yield ester products with significantly different retention times than those of the interferences of the reactive glycerol-rich matrix and in areas of the GC-chromatogram featuring lower levels of matrix interferences. A second procedure involved an initial diethyl ether/aqueous extraction of the matrix; while the extraction was found to substantially remove many of the hydrophilic matrix components and improve the overall derivatization, it also led to some loss of PA available for the derivatization. Both protocols were applied to the successful derivatization and analysis of PA by GC-MS when present at a 5 μg.mL -1 concentration in a glycerol-rich matrix sample administered during the 48th Proficiency Test administered by the Organisation for the Prohibition of Chemical Weapons (OPCW).

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Mass spectrometry and spectroscopic characterization of a tetrameric photosystem I supercomplex from Leptolyngbya ohadii , a desiccation-tolerant cyanobacterium

Cyanobacteria inhabiting desert biological soil crusts face the harsh conditions of the desert. They evolved a suite of strategies toward desiccation-hydration cycles mixed with high light irradiations, etc. In this study we purified and characterized the structure and function of Photosystem I (PSI) from Leptolyngbya ohadii, a desiccation-tolerant desert cyanobacterium. We discovered that PSI forms tetrameric (PSI-Tet) aggregate. We investigated it by using sucrose density gradient centrifugation, clear native PAGE, high performance liquid chromatography, mass spectrometry (MS), time-resolved fluorescence (TRF) and time-resolved transient absorption (TA) spectroscopy. MS analysis identified the presence of two PsaB and two PsaL proteins in PSI-Tet and uniquely revealed that PsaLs are N-terminally acetylated in contrast to non-modified PsaL in the trimeric PSI from Synechocystis sp. PCC 6803. Chlorophyll (Chl) a fluorescence decay profiles of the PSI-Tet performed at 77 K revealed two emission bands at ∼690 nm and 725 nm with the former appearing only at early delay time. The main fluorescence emission peak, associated with emission from the low energy Chls a, decays within a few nanoseconds. TA studies demonstrated that the 725 nm emission band is associated with low energy Chls a with absorption band clearly resolved at ∼710 nm at 77 K. As a result, our work suggests that the heterogenous composition of PsaBs and PsaL in PSI-Tet is related with the adaptation mechanisms needed to cope with stressful conditions under which this bacterium naturally grows.

59 BASIC BIOLOGICAL SCIENCES↗