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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Exposing structural variations in SARS-CoV-2 evolution

The mutation of SARS-CoV-2 influences viral function as residue replacements affect both physiochemical properties and folding conformations. Although a large amount of data on SARS-CoV-2 is available, the investigation of how viral functions change in response to mutations is hampered by a lack of effective structural analysis. Here, we exploit the advances of protein structure fingerprint technology to study the folding conformational changes induced by mutations. With integration of both protein sequences and folding conformations, the structures are aligned for SARS-CoV to SARS-CoV-2, including Alpha variant (lineage B.1.1.7) and Delta variant (lineage B.1.617.2). The results showed that the virus evolution with change in mutational positions and physicochemical properties increased the affinity between spike protein and ACE2, which plays a critical role in coronavirus entry into human cells. Additionally, these structural variations impact vaccine effectiveness and drug function over the course of SARS-CoV-2 evolution. The analysis of structural variations revealed how the coronavirus has gradually evolved in both structure and function and how the SARS-CoV-2 variants have contributed to more severe acute disease worldwide.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Improved bacterial recombineering by parallelized protein discovery

Exploiting bacteriophage-derived homologous recombination processes has offered precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, multiplex automated genome engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however, currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, efficient SSAPs must first be found. Here we present a high-throughput method for SSAP discovery that we call “serial enrichment for efficient recombineering” (SEER). By performing SEER in Escherichia coli to screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5- to 10-fold in E. coli , while PapRecT enables efficient recombineering in Pseudomonas aeruginosa , a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Generation of bright monomeric red fluorescent proteins via computational design of enhanced chromophore packing

Red fluorescent proteins (RFPs) have found widespread application in chemical and biological research due to their longer emission wavelengths. Here, we use computational protein design to increase the quantum yield and thereby brightness of a dim monomeric RFP (mRojoA, quantum yield = 0.02) by optimizing chromophore packing with aliphatic residues, which we hypothesized would reduce torsional motions causing non-radiative decay. Experimental characterization of the top 10 designed sequences yielded mSandy1 (λ em = 609 nm, quantum yield = 0.26), a variant with equivalent brightness to mCherry, a widely used RFP. We next used directed evolution to further increase brightness, resulting in mSandy2 (λ em = 606 nm, quantum yield = 0.35), the brightest Discosoma sp. derived monomeric RFP with an emission maximum above 600 nm reported to date. Crystallographic analysis of mSandy2 showed that the chromophore p-hydroxybenzylidene moiety is sandwiched between the side chains of Leu63 and Ile197, a structural motif that has not previously been observed in RFPs, and confirms that aliphatic packing leads to chromophore rigidification. Our results demonstrate that computational protein design can be used to generate bright monomeric RFPs, which can serve as templates for the evolution of novel far-red fluorescent proteins.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life

Abstract Modern day aerobic respiration in mitochondria involving complex I converts redox energy into chemical energy and likely evolved from a simple anaerobic system now represented by hydrogen gas-evolving hydrogenase (MBH) where protons are the terminal electron acceptor. Here we present the cryo-EM structure of an early ancestor in the evolution of complex I, the elemental sulfur (S 0 )-reducing reductase MBS. Three highly conserved protein loops linking cytoplasmic and membrane domains enable scalable energy conversion in all three complexes. MBS contains two proton pumps compared to one in MBH and likely conserves twice the energy. The structure also reveals evolutionary adaptations of MBH that enabled S 0 reduction by MBS catalyzed by a site-differentiated iron-sulfur cluster without participation of protons or amino acid residues. This is the simplest mechanism proposed for reduction of inorganic or organic disulfides. It is of fundamental significance in the iron and sulfur-rich volcanic environments of early earth and possibly the origin of life. MBS provides a new perspective on the evolution of modern-day respiratory complexes and of catalysis by biological iron-sulfur clusters.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Directed Evolution of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase Generates a Hyperactive and Highly Selective Variant

Pyrrolysyl-tRNA synthetase (PylRS) is frequently used for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. Recently, the active site of Methanomethylophilus alvus PylRS ( Ma PylRS) has been rationally engineered to expand its substrate compatibility, enabling the incorporation of difficult ncAAs. However, mutations beyond the active site that enhance the enzymatic properties of Ma PylRS have not been reported. We utilized phage-assisted non-continuous evolution (PANCE) to evolve Ma PylRS to efficiently incorporate N ε -Boc- l -lysine (BocK). Directed evolution yielded several mutations outside of the active site that greatly improve the activity of the enzyme. We combined the most effective mutations to generate a new PylRS variant (PylRS opt ) that is highly active and selective towards several lysine and phenylalanine derivatives. The mutations in PylRS opt can be used to enhance previously engineered PylRS constructs such as Ma PylRS N166S , and PylRS opt is compatible in applications requiring dual ncAA incorporation and substantially improves the yield of these target proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Polar algae flaunt their zinc assets

Metal ions have been at the centre of pivotal points in the evolution of extant life. Oxygen-evolving photosynthesis, which irrevocably reshaped the geochemistry and biology of our planet, has an absolute requirement for metal ions to harvest light, split water and transfer electrons. Access to abundant oxygen then led to the propagation of organisms capable of oxidative metabolism, a process that is also dependent on metal ions for electron transfer and reduction of molecular oxygen. Because of the essential links between metal ions and the proteins that require them to function, as well as changes in metal bioavailability through time, metals have shaped the trajectories that evolution can take. Iron typically steals this show, but with access to whole-genome sequences and transcriptomes, the imprint that zinc has made on biology is coming into focus. Writing in Nature Ecology & Evolution, Ye and colleagues present new insights into the complex connections between zinc bioavailability, adaptation of algae to the polar oceans and the evolution of regulatory networks. By combining field and laboratory-based analyses, the authors suggest that expanded families of zinc-binding proteins have enabled the green alga Microglena sp. YARC to flourish in the harsh conditions of the polar Southern Ocean (Fig. 1). As the waters where this alga occurs are known for their enrichment of zinc, the authors further propose that availability of this metal ion was directly responsible for successful microalgal colonization of polar oceans. To test this hypothesis, the authors compare meta-transcriptomes collected from pole-to-pole and find positive correlations between higher copy numbers for transcripts encoding putative zinc-binding proteins, higher latitudes, lower surface temperatures and dissolved zinc.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Differences in the dynamics of the tandem-SH2 modules of the Syk and ZAP-70 tyrosine kinases

The catalytic activity of Syk-family tyrosine kinases is regulated by a tandem Src homology 2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation that stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to phosphorylated immunoreceptor tyrosine-based activation motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP-70, that its conformation more closely resembles that of the peptide-bound state, rather than the autoinhibited state. Hydrogen–deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP-70 differ, with most of these differences occurring in the C-terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP-70 have been tuned differently, such that the autoinhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP-70, consistent with tighter control of downstream signaling pathways in T cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Discovery of an ultraspecific triuret hydrolase (TrtA) establishes the triuret biodegradation pathway

Triuret (carbonyldiurea) is an impurity found in industrial urea fertilizer (<0.1% w/w) that is applied, worldwide, around 300 million pounds each year on agricultural lands. In addition to anthropogenic sources, endogenous triuret has been identified in amoeba and human urine, the latter being diagnostic for hypokalemia. The present study is the first to describe the metabolic breakdown of triuret, which funnels into biuret metabolism. We identified the gene responsible for triuret decomposition ( trtA ) in bacterial genomes, clustered with biuH , which encodes biuret hydrolase and has close protein sequence homology. TrtA is a member of the isochorismatase-like hydrolase (IHL) protein family, similarly to BiuH, and has a catalytic efficiency (k cat/ K M ) of 6 x 10 5 M –1 s –1 , a K M for triuret of 20 μM, and exquisite substrate specificity. Indeed, TrtA has four orders of magnitude less activity with biuret. Crystal structures of TrtA in apo and holo form were solved and compared with the BiuH structure. The high substrate selectivity was found to be conveyed by second shell residues around each active site. Mutagenesis of residues conserved in TrtA to the alternate consensus found in BiuHs revealed residues critical to triuret hydrolase activity but no single mutant evolved more biuret activity, and likely a combination of mutations is required to interconvert between TrtA, BiuH functions. TrtA-mediated triuret metabolism is relatively rare in recorded genomes (1–2%), but is largely found in plant-associated, nodulating, and endophytic bacteria. This study suggests functions for triuret hydrolase in certain eukaryotic intermediary processes and prokaryotic intermediary or biodegradative metabolism.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid

Abstract We previously described the design of triacetic acid lactone (TAL) biosensor ‘AraC-TAL1’, based on the AraC regulatory protein. Although useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a ‘decoy’ ligand during negative selection, resulting in the isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the iterative protein redesign and optimization computational framework was employed, followed by 2 rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.

Wang, Zhiqing↗
DOE OSTI2020

Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study

Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today’s agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized more quickly than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cell’s growth rate to the target gene’s function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Epistasis and evolution: recent advances and an outlook for prediction

As organisms evolve, the effects of mutations change as a result of epistatic interactions with other mutations accumulated along the line of descent. This can lead to shifts in adaptability or robustness that ultimately shape subsequent evolution. Here, we review recent advances in measuring, modeling, and predicting epistasis along evolutionary trajectories, both in microbial cells and single proteins. We focus on simple patterns of global epistasis that emerge in this data, in which the effects of mutations can be predicted by a small number of variables. The emergence of these patterns offers promise for efforts to model epistasis and predict evolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization

There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli : methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13 C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.

Bennett, R. Kyle↗
DOE OSTI2022

Photocatalytic Hydrogen Evolution by a De Novo Designed Metalloprotein that Undergoes Ni–Mediated Oligomerization Shift

De novo metalloprotein design involves the construction of proteins guided by specific repeat patterns of polar and apolar residues, which, upon self-assembly, provide a suitable environment to bind metals and produce artificial metalloenzymes. While a wide range of functionalities have been realized in de novo designed metalloproteins, the functional repertoire of such constructs towards alternative energy-relevant catalysis is currently limited. Here we show the application of de novo approach to design a functional H 2 evolving protein. The design involved the assembly of an amphiphilic peptide featuring cysteines at tandem a/d sites of each helix. Intriguingly, upon Ni II addition, the oligomers shift from a major trimeric assembly to a mix of dimers and trimers. The metalloprotein produced H 2 photocatalytically with a bell-shape pH dependence, having a maximum activity at pH 5.5. Transient absorption spectroscopy is used to determine the timescales of electron transfer as a function of pH. Selective outer sphere mutations are made to probe how the local environment tunes activity. Finally, a preferential enhancement of activity is observed via steric modulation above the Ni II site, towards the N-termini, compared to below the Ni II site towards the C-termini.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea

Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Optimizing immunization protocols to elicit broadly neutralizing antibodies

Significance The global health burden could be substantially alleviated by the creation of universal vaccines against highly mutable pathogens like HIV and influenza. Broadly neutralizing antibodies (bnAbs) are encouraging targets for such vaccines, because they can bind to diverse strains of these pathogens. BnAbs typically develop only after the immune system has been exposed to many mutated versions of a pathogen, and then too in few people and low numbers. Thus, sequentially administering multiple different pathogen-like proteins is a promising strategy to elicit bnAbs through vaccination. However, it remains unclear how best to design/administer these proteins. We explore this using physics-based simulations and provide mechanistic insights into antibody evolution that could guide creation of universal vaccines against highly mutable pathogens.

Sprenger, Kayla G.↗
DOE OSTI2024

Rapid discovery and evolution of nanosensors containing fluorogenic amino acids

Binding-activated optical sensors are powerful tools for imaging, diagnostics, and biomolecular sensing. However, biosensor discovery is slow and requires tedious steps in rational design, screening, and characterization. Here we report on a platform that streamlines biosensor discovery and unlocks directed nanosensor evolution through genetically encodable fluorogenic amino acids (FgAAs). Building on the classical knowledge-based semisynthetic approach, we engineer ~15 kDa nanosensors that recognize specific proteins, peptides, and small molecules with up to 100-fold fluorescence increases and subsecond kinetics, allowing real-time and wash-free target sensing and live-cell bioimaging. An optimized genetic code expansion chemistry with FgAAs further enables rapid (~3 h) ribosomal nanosensor discovery via the cell-free translation of hundreds of candidates in parallel and directed nanosensor evolution with improved variant-specific sensitivities (up to ~250-fold) for SARS-CoV-2 antigens. Altogether, this platform could accelerate the discovery of fluorogenic nanosensors and pave the way to modify proteins with other non-standard functionalities for diverse applications.

Biosensors↗
DOE OSTI2024

Cytochrome b5 diversity in green lineages preceded the evolution of syringyl lignin biosynthesis

Abstract Lignin production marked a milestone in vascular plant evolution, and the emergence of syringyl (S) lignin is lineage specific. S-lignin biosynthesis in angiosperms, mediated by ferulate 5-hydroxylase (F5H, CYP84A1), has been considered a recent evolutionary event. F5H uniquely requires the cytochrome b5 protein CB5D as an obligatory redox partner for catalysis. However, it remains unclear how CB5D functionality originated and whether it coevolved with F5H. We reveal here the ancient evolution of CB5D-type function supporting F5H-catalyzed S-lignin biosynthesis. CB5D emerged in charophyte algae, the closest relatives of land plants, and is conserved and proliferated in embryophytes, especially in angiosperms, suggesting functional diversification of the CB5 family before terrestrialization. A sequence motif containing acidic amino residues in Helix 5 of the CB5 heme-binding domain contributes to the retention of CB5D function in land plants but not in algae. Notably, CB5s in the S-lignin–producing lycophyte Selaginella lack these residues, resulting in no CB5D-type function. An independently evolved S-lignin biosynthetic F5H (CYP788A1) in Selaginella relies on NADPH-dependent cytochrome P450 reductase as sole redox partner, distinct from angiosperms. These results suggest that angiosperm F5Hs coopted the ancient CB5D, forming a modern cytochrome P450 monooxygenase system for aromatic ring meta-hydroxylation, enabling the reemergence of S-lignin biosynthesis in angiosperms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Generation of Pseudomonas putida KT2440 Strains with Efficient Utilization of Xylose and Galactose via Adaptive Laboratory Evolution

While Pseudomonas putida KT2440 has great potential for biomass-converting processes, its inability to utilize the biomass abundant sugars xylose and galactose has limited its applications. Here, in this study, we utilized Adaptive Laboratory Evolution (ALE) to optimize engineered KT2440 with heterologous expression of xylD encoding xylonate dehydratase from Caulobacter crescentus and galETKM encoding UDP-glucose 4-epimerase, galactose-1-phosphate uridylyltransferase, galactokinase, and galactose-1-epimerase from Escherichia coli K-12 MG1655. Poor starting strain growth (<0.1 h –1 or none) was evolutionarily optimized to rates of up to 0.25 h –1 on xylose and 0.52 h –1 on galactose. Whole-genome sequencing, transcriptomic analysis, and growth screens revealed significant roles of kguT encoding a 2-ketogluconate operon repressor and 2-ketogluconate transporter, and gtsABCD encoding an ATP-binding cassette (ABC) sugar transporting system in xylose and galactose growth conditions, respectively. Finally, we expressed the heterologous indigoidine production pathway in the evolved and unevolved engineered strains and successfully produced 3.2 g/L and 2.2 g/L from 10 g/L of either xylose or galactose in the evolved strains whereas the unevolved strains did not produce any detectable product. Thus, the generated KT2440 strains have the potential for broad application as optimized platform chassis to develop efficient microorganism-based biomass-utilizing bioprocesses.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗