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Results for “protein acetylation”

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NASA NTRS1995

Determination of (alpha)-dialkylamino acids and their Enantiomers in Geological Samples by High-Performance Liquid Chromatography after Dervatization with a Chiral Adduct of (omicron)-Phthaldialdehyde

Derivatization with (omicron)-phthaldialdehyde (OPA) and the chiral thiol N-acetyl-L-cysteine (NAC) is a convenient and sensitive technique for the HPLC detection and resolution of protein amino acid enantiomers. The kinetics of the reaction of OPA-NAC with (alpha)-dialkylamino acids was investigated. The fluorescence yield of (alpha)-dialkylamino acids was only about 10% of that of protein amino acids when the derivatization was carried out at room temperature for 1-2 min, which is the procedure generally used for protein amino acid analyses. The fluorescence yield of (alpha)-dialkylamino acids can be enhanced by up to ten-fold when the derivatization reaction time is increased to 15 min at room temperature. The OPA-NAC technique was optimized for the detection and enantiomeric resolution of a-dialkylamino acids in geological samples which contain a large excess of protein amino acids. The estimated detection limit for a-dialkylamino acids is 1-2 pmol, comparable to that for protein amino acids.

Zhoa, Meixun↗

DOE OSTI2022

Thioester synthesis by a designed nickel enzyme models prebiotic energy conversion

The formation of carbon–carbon bonds from prebiotic precursors such as carbon dioxide represents the foundation of all primordial life processes. In extant organisms, this reaction is carried out by the carbon monoxide dehydrogenase (CODH)/acetyl coenzyme A synthase (ACS) enzyme, which performs the cornerstone reaction in the ancient Wood–Ljungdahl metabolic pathway to synthesize the key biological metabolite, acetyl-CoA. Despite its significance, a fundamental understanding of this transformation is lacking, hampering efforts to harness analogous chemistry. To address these knowledge gaps, we have designed an artificial metalloenzyme within the azurin protein scaffold as a structural, functional, and mechanistic model of ACS. We demonstrate the intermediacy of the Ni I species and requirement for ordered substrate binding in the bioorganometallic carbon–carbon bond-forming reaction from the one-carbon ACS substrates. The electronic and geometric structures of the nickel-acetyl intermediate have been characterized using time-resolved optical, electron paramagnetic resonance, and X-ray absorption spectroscopy in conjunction with quantum chemical calculations. Moreover, we demonstrate that the nickel-acetyl species is chemically competent for selective acyl transfer upon thiol addition to biosynthesize an activated thioester. Drawing an analogy to the native enzyme, a mechanism for thioester generation by this ACS model has been proposed. The fundamental insight into the enzymatic process provided by this rudimentary ACS model has implications for the evolution of primitive ACS-like proteins. Ultimately, these findings offer strategies for development of highly active catalysts for sustainable generation of liquid fuels from one-carbon substrates, with potential for broad applications across diverse fields ranging from energy storage to environmental remediation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2025

Proteome-wide characterization of PTMs reveals host cell responses to viral infection and identifies putative antiviral drug targets

Post-translational modifications (PTMs) are biochemical modifications that can significantly alter protein structure, function, stability, localization, and interactions with other molecules, thereby activating or inactivating intracellular processes. A growing body of research has begun to highlight the role of PTMs, including phosphorylation, ubiquitination, acetylation, and redox modifications, during virus-host interactions. Collectively, these PTMs regulate key steps in mounting the host immune response and control critical host pathways required for productive viral replication. This has led to the conception of antiviral therapeutics that focus on controlling host protein PTMs, potentially offering pathogen-agnostic treatment options and revolutionizing our capacity to prevent virus transmission. On the other hand, viruses can hijack the host cellular PTM machinery to modify viral proteins in promoting viral replication and evading immune surveillance. PTM regulation during virus-host interactions is complex and poorly mapped, and the development of effective PTM-targeted antiviral drugs will require a more comprehensive understanding of the cellular pathways essential for virus replication. In this review, we discuss the roles of PTMs in virus infection and how technological advances in mass spectrometry-based proteomics can capture systems-level PTM changes during viral infection. Additionally, we explore how such knowledge is leveraged to identify PTM-targeted candidates for developing antiviral drugs. Looking ahead, studies focusing on the discovery and functional elucidation of PTMs, either on the host or viral proteins, will not only deepen our understanding of molecular pathology but also pave the way for developing better drugs to fight emerging viruses.

Immunology↗

DOE OSTI2022

Chromatin Changes in Phytochrome Interacting Factor-Regulated Genes Parallel Their Rapid Transcriptional Response to Light

As sessile organisms, plants must adapt to a changing environment, sensing variations in resource availability and modifying their development in response. Light is one of the most important resources for plants, and its perception by sensory photoreceptors (e.g., phytochromes) and subsequent transduction into long-term transcriptional reprogramming have been well characterized. Chromatin changes have been shown to be involved in photomorphogenesis. However, the initial short-term transcriptional changes produced by light and what factors enable these rapid changes are not well studied. Here, we define rapidly light-responsive, Phytochrome Interacting Factor (PIF) direct-target genes (LRP-DTGs). We found that a majority of these genes also show rapid changes in Histone 3 Lysine-9 acetylation (H3K9ac) in response to the light signal. Detailed time-course analysis of transcript and chromatin changes showed that, for light-repressed genes, H3K9 deacetylation parallels light-triggered transcriptional repression, while for light-induced genes, H3K9 acetylation appeared to somewhat precede light-activated transcript accumulation. However, direct, real-time imaging of transcript elongation in the nucleus revealed that, in fact, transcriptional induction actually parallels H3K9 acetylation. Collectively, the data raise the possibility that light-induced transcriptional and chromatin-remodeling processes are mechanistically intertwined. Histone modifying proteins involved in long term light responses do not seem to have a role in this fast response, indicating that different factors might act at different stages of the light response. This work not only advances our understanding of plant responses to light, but also unveils a system in which rapid chromatin changes in reaction to an external signal can be studied under natural conditions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Characterization of a novel aromatic substrate-processing microcompartment in Actinobacteria

ABSTRACT We have discovered a new cluster of genes that is found exclusively in the Actinobacteria phylum. This locus includes genes for the 2-aminophenol meta -cleavage pathway and the shell proteins of a bacterial microcompartment (BMC) and has been named aromatics (ARO) for its putative role in the breakdown of aromatic compounds. In this study, we provide details about the distribution and composition of the ARO BMC locus and conduct phylogenetic, structural, and functional analyses of the first two enzymes in the catabolic pathway: a unique 2-aminophenol dioxygenase, which is exclusively found alongside BMC shell genes in Actinobacteria, and a semialdehyde dehydrogenase, which works downstream of the dioxygenase. Genomic analysis reveals variations in the complexity of the ARO loci across different orders. Some loci are simple, containing shell proteins and enzymes for the initial steps of the catabolic pathway, while others are extensive, encompassing all the necessary genes for the complete breakdown of 2-aminophenol into pyruvate and acetyl-CoA. Furthermore, our analysis uncovers two subtypes of ARO BMC that likely degrade either 2-aminophenol or catechol, depending on the presence of a pathway-specific gene within the ARO locus. The precise precursor of 2-aminophenol, which serves as the initial substrate and/or inducer for the ARO pathway, remains unknown, as our model organism Micromonospora rosaria cannot utilize 2-aminophenol as its sole energy source. However, using enzymatic assays, we demonstrate the dioxygenase’s ability to cleave both 2-aminophenol and catechol in vitro , in collaboration with the aldehyde dehydrogenase, to facilitate the rapid conversion of these unstable and toxic intermediates. IMPORTANCE Bacterial microcompartments (BMCs) are proteinaceous organelles that are widespread among bacteria and provide a competitive advantage in specific environmental niches. Studies have shown that the genetic information necessary to form functional BMCs is encoded in loci that contain genes encoding shell proteins and the enzymatic core. This allows the bioinformatic discovery of BMCs with novel functions and expands our understanding of the metabolic diversity of BMCs. ARO loci, found only in Actinobacteria, contain genes encoding for phylogenetically remote shell proteins and homologs of the meta -cleavage degradation pathway enzymes that were shown to convert central aromatic intermediates into pyruvate and acetyl-CoA in gamma Proteobacteria. By analyzing the gene composition of ARO BMC loci and characterizing two core enzymes phylogenetically, structurally, and functionally, we provide an initial functional characterization of the ARO BMC, the most unusual BMC identified to date, distinctive among the repertoire of studied BMCs.

2-AP 1,6-dioxygenase↗

NASA NTRS1992

Preferential hydrophobic interactions are responsible for a preference of D-amino acids in the aminoacylation of 5'-AMP with hydrophobic amino acids

We have studied the chemistry of aminoacyl AMP to model reactions at the 3' terminus of aminoacyl tRNA for the purpose of understanding the origin of protein synthesis. The present studies relate to the D, L preference in the esterification of 5'-AMP. All N-acetyl amino acids we studied showed faster reaction of the D-isomer, with a generally decreasing preference for D-isomer as the hydrophobicity of the amino acid decreased. The beta-branched amino acids, Ile and Val, showed an extreme preference for D-isomer. Ac-Leu, the gamma-branched amino acid, showed a slightly low D/L ratio relative to its hydrophobicity. The molecular basis for these preferences for D-isomer is understandable in the light of our previous studies and seems to be due to preferential hydrophobic interaction of the D-isomer with adenine. The preference for hydrophobic D-amino acids can be decreased by addition of an organic solvent to the reaction medium. Conversely, peptidylation with Ac-PhePhe shows a preference for the LL isomer over the DD isomer.

Non-NASA Center↗

DOE OSTI2023

Cryo-EM structure of the human Sirtuin 6–nucleosome complex

Sirtuin 6 (SIRT6) is a multifaceted protein deacetylase/deacylase and a major target for small-molecule modulators of longevity and cancer. In the context of chromatin, SIRT6 removes acetyl groups from histone H3 in nucleosomes, but the molecular basis for its nucleosomal substrate preference is unknown. Our cryo–electron microscopy structure of human SIRT6 in complex with the nucleosome shows that the catalytic domain of SIRT6 pries DNA from the nucleosomal entry-exit site and exposes the histone H3 N-terminal helix, while the SIRT6 zinc-binding domain binds to the histone acidic patch using an arginine anchor. In addition, SIRT6 forms an inhibitory interaction with the C-terminal tail of histone H2A. The structure provides insights into how SIRT6 can deacetylate both H3 K9 and H3 K56.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1993

N-Acetyl-D- and L-esters of 5'-AMP hydrolyze at different rates

Studies of the properties of aminoacyl derivatives of 5'-AMP are aimed at understanding the origin of the process of protein synthesis. Aminoacyl (2',3') esters of 5'-AMP can serve as models of the 3'-terminus of aminoacyl tRNA. We report here on the relative rates of hydrolysis of Ac-D- and L-Phe AMP esters as a function of pH. At all pHs above 3, the rate constant of hydrolysis of the Ac-L-Phe ester is 1.7 to 2.1 times that of Ac-D-Phe ester. The D-isomer seems partially protected from hydrolysis by a stronger association with the adenine ring of the 5'-AMP.

Non-NASA Center↗

DOE OSTI2023

An Immunomodulatory Polysaccharide–Protein Complex Isolated from the Polypore Fungus Royoporus badius

Many wild edible polypore mushrooms have medicinal value. In this study, we investigate the potential medicinal properties of the wild polypore mushroom Royoporus badius collected from north-central British Columbia, Canada. Water extract from R. badius was found to exhibit potent immunomodulatory activity. The extract was purified using DEAE-Sephadex anion-exchange chromatography as well as Sephacryl S-500 and HPLC BioSEC5 size-exclusion chromatography, to yield a novel polysaccharide-protein complex (IMPP-Rb).IMPP-Rb has a peak maxima molecular weight (M p ) of 950 kDa. GC-MS analyses showed that IMPP-Rb is composed predominantly of glucose (49.2%), galactose (11.3%), mannose (10.8%), rhamnose (9.6%), and galacturonic acid (8.2%), with smaller amounts of xylose (5.2%), fucose (2.8%), N-acetyl glucosamine (1.8%), and arabinose (1.2%). IMPP-Rb has multiple linkages, with 4-Glcp, 4-Manp, 6-Manp, 3,4-Manp, 4-Xylp, and 2-Rhap being the most prominent. IMPP-Rb is capable of inducing many cytokines in vitro and the protein component is indispensable for its immunomodulatory activity. IMPP-Rb has potential application as an immuno-stimulatory agent with pharmaceutical value.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Selective Inhibitors with Cellular Target Engagement**

Abstract Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, especially cancer. Five HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc‐dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement, e. g., in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of most other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like acetyl‐putrescine or ‐spermidine. Hence, it is also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin‐labelled acetyl‐spermidine derivative to measure its PDAC activity, which is suitable for high‐throughput screening. Using this assay, we identified potent inhibitors of HDAC10‐mediated spermidine deacetylation in vitro. Based on the oligoamine preference of HDAC10, we also designed inhibitors with a basic moiety in appropriate distance to the zinc binding hydroxamate that showed potent inhibition of HDAC10 with high selectivity, and we solved a HDAC10‐inhibitor structure using X‐ray crystallography. We could demonstrate selective cellular target engagement for HDAC10 but a lysosomal phenotype in neuroblastoma cells that was previously associated with HDAC10 inhibition was not observed. Thus, we have developed new chemical probes for HDAC10 that allow further clarification of the biological role of this enzyme.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2025

Determination of Thiol Protonation States by Sulfur X-ray Spectroscopy in Biological Systems

Cysteine is one of the most functionally diverse of the proteinogenic amino acids, owing to its reactive thiol side chain that can undergo deprotonation to form a strongly nucleophilic thiolate. However, few techniques can directly interrogate sulfur charge and covalency in cysteine, particularly in proteins. X-ray spectroscopies provide an element specific probe of sulfur. We demonstrate the sensitivity of S Kβ and Kα X-ray emission spectroscopy (XES) to cysteine ionization and compare it to S K-edge X-ray absorption spectroscopy (XAS) in the physiologically relevant biomolecules l-cysteine and N-acetyl-l-cysteine at room temperature in solution phase. Kβ XES and K-edge XAS are most sensitive to chemical changes at the cysteine thiol and can be used to evaluate the composition of thiol/thiolate mixtures. Furthermore, these results provide a foundation for assessing the pK a of functionally significant cysteine residues in proteins and open the door to time-resolved studies of cysteine-dependent enzymes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

HNRNPU promotes the progression of hepatocellular carcinoma by enhancing CDK2 transcription

Highlights: • The expression of HNRNPU is upregulated in hepatocellular carcinoma. • HNRNPU knockdown significantly inhibits HCC cell proliferation. • HNRNPU knockdown induces cell cycle arrest. • HNRNPU regulates CDK2 transcription by directly binding to the CDK2 gene locus. The nuclear matrix-associated protein Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU), also known as SAF-A, is known to maintain active chromatin structure in mouse hepatocytes. However, the functional roles and molecular mechanisms of HNRNPU in the development of hepatocellular carcinoma (HCC) remain largely unknown. Herein, we found that HNRNPU was upregulated in HCC, and the proliferation of HCC cells was inhibited in vitro and in vivo upon HNRNPU knockdown. Moreover, the upregulation of HNRNPU was correlated with poor prognosis in HCC. Mechanistically, HNRNPU bound to the CDK2 gene locus, a key factor in cell cycle regulation, where it was enriched with H3K27 acetylation (H3K27ac), H3K9 acetylation (H3K9ac), and H3K4 mono-methylation (H3K4me1). Furthermore, HNRNPU knockdown reduced the levels of H3K27ac and H3K9ac at the binding site, where the levels of H3K27 tri-methylation (H3K27me3) were increased, eventually leading to the downregulation of CDK2. Collectively, our results provide a new mechanism whereby HNRNPU promotes HCC development by enhancing the transcription of CDK2.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2001

Fabrication of poly(ethylene glycol) hydrogel microstructures using photolithography

The fabrication of hydrogel microstructures based upon poly(ethylene glycol) diacrylates, dimethacrylates, and tetraacrylates patterned photolithographically on silicon or glass substrates is described. A silicon/silicon dioxide surface was treated with 3-(trichlorosilyl)propyl methacrylate to form a self-assembled monolayer (SAM) with pendant acrylate groups. The SAM presence on the surface was verified using ellipsometry and time-of-flight secondary ion mass spectrometry. A solution containing an acrylated or methacrylated poly(ethylene glycol) derivative and a photoinitiator (2,2-dimethoxy-2-phenylacetophenone) was spin-coated onto the treated substrate, exposed to 365 nm ultraviolet light through a photomask, and developed with either toluene, water, or supercritical CO2. As a result of this process, three-dimensional, cross-linked PEG hydrogel microstructures were immobilized on the surface. Diameters of cylindrical array members were varied from 600 to 7 micrometers by the use of different photomasks, while height varied from 3 to 12 micrometers, depending on the molecular weight of the PEG macromer. In the case of 7 micrometers diameter elements, as many as 400 elements were reproducibly generated in a 1 mm2 square pattern. The resultant hydrogel patterns were hydrated for as long as 3 weeks without delamination from the substrate. In addition, micropatterning of different molecular weights of PEG was demonstrated. Arrays of hydrogel disks containing an immobilized protein conjugated to a pH sensitive fluorophore were also prepared. The pH sensitivity of the gel-immobilized dye was similar to that in an aqueous buffer, and no leaching of the dye-labeled protein from the hydrogel microstructure was observed over a 1 week period. Changes in fluorescence were also observed for immobilized fluorophore labeled acetylcholine esterase upon the addition of acetyl acholine.

NASA Discipline Life Sciences Technologies↗

DOE OSTI2021

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↗

DOE OSTI2023

Spray‐induced gene silencing to identify powdery mildew gene targets and processes for powdery mildew control

Abstract Spray‐induced gene silencing (SIGS) is an emerging tool for crop pest protection. It utilizes exogenously applied double‐stranded RNA to specifically reduce pest target gene expression using endogenous RNA interference machinery. In this study, SIGS methods were developed and optimized for powdery mildew fungi, which are widespread obligate biotrophic fungi that infect agricultural crops, using the known azole‐fungicide target cytochrome P450 51 (CYP51) in the Golovinomyces orontii–Arabidopsis thaliana pathosystem. Additional screening resulted in the identification of conserved gene targets and processes important to powdery mildew proliferation: apoptosis‐antagonizing transcription factor in essential cellular metabolism and stress response; lipid catabolism genes lipase a , lipase 1 , and acetyl‐CoA oxidase in energy production ; and genes involved in manipulation of the plant host via abscisic acid metabolism ( 9‐cis‐epoxycarotenoid dioxygenase , xanthoxin dehydrogenase , and a putative abscisic acid G‐protein coupled receptor ) and secretion of the effector protein, effector candidate 2 . Powdery mildew is the dominant disease impacting grapes and extensive powdery mildew resistance to applied fungicides has been reported. We therefore developed SIGS for the Erysiphe necator–Vitis vinifera system and tested six successful targets identified using the G. orontii–A. thaliana system. For all targets tested, a similar reduction in powdery mildew disease was observed between systems. This indicates screening of broadly conserved targets in the G. orontii–A. thaliana pathosystem identifies targets and processes for the successful control of other powdery mildew fungi. The efficacy of SIGS on powdery mildew fungi makes SIGS an exciting prospect for commercial powdery mildew control.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2003

Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-Dependent Enzymes Revealed by Human Pyruvate Dehydrogenase

Thiamin pyrophosphate (TPP), the biologically active form of vitamin BI, is a cofactor of enzymes catalyzing reactions involving the cleavage of a carbon-carbon bond adjacent to an oxo group. TPP-dependent enzymes show a common mechanism of TPP activation by: (1) forming the ionic N-H...O(sup -) hydrogen bonding between the N1' atom of the aminopirymidine ring of the coenzyme and intrinsic gamma-carboxylate group of glutamate and (2) imposing an "active" V-conformation that brings the N4' atom of the aminopirymidine to the distance required for the intramolecular C-H.. .N hydrogen bonding with the thiazolium C2 atom. Within these two hydrogen bonds that rapidly exchange protons, protonation of the N1' atom is strictly coordinated with the deprotonation of the 4' -amino group and eventually abstraction of the proton from C2. The human pyruvate dehydrogenase Elp, component of human pyruvate dehydrogenase complex, catalyzes the irreversible decarboxylation of the pyruvate followed by the reductive acetylation of the lipoyl group of dihydrolipoyl acyltransferase. Elp is alpha(sub 2)beta(sub2)-heterotetrameric with a molecular mass of I54 kDa, which has two catalytic sites, each providing TPP and magnesium ion as cofactors and each formed on the interface between the PP and PYR domains. The dynamic nonequivalence of two otherwise chemically equivalent catalytic sites has been observed and the flip-flop mechanism was suggested, according to which two active sites affect each other and in which different steps of the catalytic reaction are performed in each of the sites at any given moment. Based on specific futures of human pyruvate dehydrogenase including rigid and flexible connections between domains that bind the cofactor we propose a mechanistic model for the flip-flop action of this enzyme. We postulate that the dynamic protein environment drives the exchange of tautomers in the 4' -aminopyrimidine ring of the cofactor through a concerted shuttl-like motion of tightly connected domains. The dynamic exchange of those tautomers, in turns, is required during the reactions of pyruvate decarboxylation and reductive acetylation of lipoamide. Thus the shuttle-like motion of the domains is coordinated with the reactions of decarboxylation and acetylation, which are carried out in each of the cofactor sites resulting in a flip-flop action of the enzyme. The structure-derived mechanism of action of human pyruvate dehydrogenase may be likely common for other TPP-dependent enzymes.

Dominiak, Paulina↗

DOE OSTI2026

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↗

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↗