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At least 541 records · Page 30
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Indirect Routes to Aminoacyl-tRNA: The Diversity of Prokaryotic Cysteine Encoding Systems

Universally present aminoacyl-tRNA synthetases (aaRSs) stringently recognize their cognate tRNAs and acylate them with one of the proteinogenic amino acids. However, some organisms possess aaRSs that deviate from the accurate translation of the genetic code and exhibit relaxed specificity toward their tRNA and/or amino acid substrates. Typically, these aaRSs are part of an indirect pathway in which multiple enzymes participate in the formation of the correct aminoacyl-tRNA product. The indirect cysteine (Cys)-tRNA pathway, originally thought to be restricted to methanogenic archaea, uses the unique O -phosphoseryl-tRNA synthetase (SepRS), which acylates the non-proteinogenic amino acid O -phosphoserine (Sep) onto tRNA Cys . Together with Sep-tRNA:Cys-tRNA synthase (SepCysS) and the adapter protein SepCysE, SepRS forms a transsulfursome complex responsible for shuttling Sep-tRNA Cys to SepCysS for conversion of the tRNA-bound Sep to Cys. Here, we report a comprehensive bioinformatic analysis of the diversity of indirect Cys encoding systems. These systems are present in more diverse groups of bacteria and archaea than previously known. Given the occurrence and distribution of some genes consistently flanking SepRS, it is likely that this gene was part of an ancient operon that suffered a gradual loss of its original components. Newly identified bacterial SepRS sequences strengthen the suggestion that this lineage of enzymes may not rely on the m 1 G37 identity determinant in tRNA. Some bacterial SepRSs possess an N-terminal fusion resembling a threonyl-tRNA synthetase editing domain, which interestingly is frequently observed in the vicinity of archaeal SepCysS genes. We also found several highly degenerate SepRS genes that likely have altered amino acid specificity. Cross-analysis of selenocysteine (Sec)-utilizing traits confirmed the co-occurrence of SepCysE and the Sec-utilizing machinery in archaea, but also identified an unusual O -phosphoseryl-tRNA Sec kinase fusion with an archaeal Sec elongation factor in some lineages, where it may serve in place of SepCysE to prevent crosstalk between the two minor aminoacylation systems. These results shed new light on the variations in SepRS and SepCysS enzymes that may reflect adaptation to lifestyle and habitat, and provide new information on the evolution of the genetic code.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Identification of recG genetic interactions in Escherichia coli by transposon sequencing

ABSTRACT Maintaining the integrity of the genome is of utmost importance for cell division and propagation. In Escherichia coli , the RecG protein has been implicated in processing branched recombination intermediates during DNA repair processes, but the primary cellular role(s) of RecG and the repair pathways in which it acts have been difficult to define. To gain additional insight into RecG function, we employed transposon sequencing to identify recG genetic interactions and reveal complementary or redundant functions. The strongest hits from the screen were the dam , uvrD , rnhA , radA , and rep genes. The conditional importance of these five genes in cells lacking recG was confirmed using a plasmid-based assay, revealing synthetic lethal interactions for most double deletion strains. Several of the synthetic lethal gene combinations (with uvrD, rep, and radA , but not rnhA or dam ) were suppressed by deletion of recF or recO , indicating that their genetic relationships involved roles in post-replication gap repair. Additionally, loss of the RecG/SSB interaction phenocopied a recG deletion when combined with dam , uvrD , radA , or rnhA deletions but not with rep . The results reinforce the idea of RecG as a general genome guardian. RecG has at least two functions. It plays an important role in the resolution of joint molecules behind the fork, formed during post-replication gap repair. RecG is also required to suppress genome over-replication caused by unscheduled replication initiation at R-loops, at double-strand breaks caused by dam inactivation, and during replication termination. IMPORTANCE DNA damage and subsequent DNA repair processes are mutagenic in nature and an important driver of evolution in prokaryotes, including antibiotic resistance development. Genetic screening approaches, such as transposon sequencing (Tn-seq), have provided important new insights into gene function and genetic relationships. Here, we employed Tn-seq to gain insight into the function of the recG gene, which renders Escherichia coli cells moderately sensitive to a variety of DNA-damaging agents when they are absent. The reported recG genetic interactions can be used in combination with future screens to aid in a more complete reconstruction of DNA repair pathways in bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1996

Homochirality as the signature of life: the SETH Cigar

A characteristic hallmark of life is its homochirality: all biomolecules are usually of one hand, e.g. on Earth life uses only L-amino acids for protein synthesis and not their D mirror images. It is therefore suggested that a search for extra-terrestrial life can be approached as a Search for Extra-Terrestrial Homochirality (SETH). A novel miniaturized space polarimeter, called the SETH Cigar, is described which could he used to detect optical rotation as the homochiral signature of life on other planets. Moving parts are avoided by replacing the normal rotating polarizer by multiple fixed polarizers at different angles as in the eye of the bee. It is believed that homochirality will be found in the subsurface layers on Mars as a relic of extinct life.

NASA Center ARC↗
DOE OSTI2021

Plant ecological genomics at the limits of life in the Atacama Desert

The Atacama Desert in Chile—hyperarid and with high–ultraviolet irradiance levels—is one of the harshest environments on Earth. Yet, dozens of species grow there, including Atacama-endemic plants. Herein, we establish the Talabre–Lejía transect (TLT) in the Atacama as an unparalleled natural laboratory to study plant adaptation to extreme environmental conditions. We characterized climate, soil, plant, and soil–microbe diversity at 22 sites (every 100 m of altitude) along the TLT over a 10-y period. We quantified drought, nutrient deficiencies, large diurnal temperature oscillations, and pH gradients that define three distinct vegetational belts along the altitudinal cline. Further, we deep-sequenced transcriptomes of 32 dominant plant species spanning the major plant clades, and assessed soil microbes by metabarcoding sequencing. The top-expressed genes in the 32 Atacama species are enriched in stress responses, metabolism, and energy production. Moreover, their root-associated soils are enriched in growth-promoting bacteria, including nitrogen fixers. To identify genes associated with plant adaptation to harsh environments, we compared 32 Atacama species with the 32 closest sequenced species, comprising 70 taxa and 1,686,950 proteins. To perform phylogenomic reconstruction, we concatenated 15,972 ortholog groups into a supermatrix of 8,599,764 amino acids. Using two codon-based methods, we identified 265 candidate positively selected genes (PSGs) in the Atacama plants, 64% of which are located in Pfam domains, supporting their functional relevance. For 59/184 PSGs with an Arabidopsis ortholog, we uncovered functional evidence linking them to plant resilience. As some Atacama plants are closely related to staple crops, these candidate PSGs are a “genetic goldmine” to engineer crop resilience to face climate change.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Molecular basis of P[II] major human rotavirus VP8* domain recognition of histo-blood group antigens

Initial cell attachment of rotavirus (RV) to specific cell surface glycan receptors, which is the essential first step in RV infection, is mediated by the VP8* domain of the spike protein VP4. Recently, human histo-blood group antigens (HBGAs) have been identified as receptors or attachment factors for human RV strains. RV strains in the P[4] and P[8] genotypes of the P[II] genogroup share common recognition of the Lewis b (Le b ) and H type 1 antigens, however, the molecular basis of receptor recognition by the major human P[8] RVs remains unknown due to lack of experimental structural information. Here, we used nuclear magnetic resonance (NMR) spectroscopy-based titration experiments and NMR-derived high ambiguity driven docking (HADDOCK) methods to elucidate the molecular basis for P[8] VP8* recognition of the Leb (LNDFH I) and type 1 HBGAs. We also used X-ray crystallography to determine the molecular details underlying P[6] recognition of H type 1 HBGAs. Unlike P[6]/P[19] VP8*s that recognize H type 1 HBGAs in a binding surface composed of an α-helix and a β-sheet, referred as the “βα binding site”, the P[8] and P[4] VP8*s bind Le b HBGAs in a previously undescribed pocket formed by the edges of two β-sheets, referred to as the “ββ binding site”. Importantly, the P[8] and P[4] VP8*s retain binding capability to non-Le b type 1 HBGAs using the βα binding site. The presence of two distinct binding sites for Le b and non-Le b HBGA glycans in the P[8] and P[4] VP8* domains suggests host-pathogen co-evolution under structural and functional adaptation of RV pathogens to host glycan polymorphisms. Assessment and understanding of the precise impact of this co-evolutionary process in determining RV host ranges and cross-species RV transmission should facilitate improved RV vaccine development and prediction of future RV strain emergence and epidemics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The Moderately (D)efficient Enzyme: Catalysis-Related Damage In Vivo and Its Repair

Enzymes have in vivo life spans. Analysis of life spans, i.e., lifetime totals of catalytic turnovers, suggests that nonsurvivable collateral chemical damage from the very reactions that enzymes catalyze is a common but underdiagnosed cause of enzyme death. Analysis also implies that many enzymes are moderately deficient in that their active-site regions are not naturally as hardened against such collateral damage as they could be, leaving room for improvement by rational design or directed evolution. Enzyme life span might also be improved by engineering systems that repair otherwise fatal active-site damage, of which a handful are known and more are inferred to exist. Unfortunately, the data needed to design and execute such improvements are lacking: there are too few measurements of in vivo life span, and existing information about the extent, nature, and mechanisms of active-site damage and repair during normal enzyme operation is too scarce, anecdotal, and speculative to act on. Fortunately, advances in proteomics, metabolomics, cheminformatics, comparative genomics, and structural biochemistry now empower a systematic, data-driven approach for identifying, predicting, and validating instances of active-site damage and its repair. These capabilities would be practically useful in enzyme redesign and improvement of in-use stability and could change our thinking about which enzymes die young in vivo, and why.

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

National Cancer Institute Think-Tank Meeting Report on Proteomic Cartography and Biomarkers at the Single-Cell Level: Interrogation of Premalignant Lesions

A Think-Tank Meeting was convened by the National Cancer Institute (NCI) to solicit experts’ opinion on the development and application of multi-omic single-cell analyses, and especially single-cell proteomics, to improve the development of a new generation of biomarkers for cancer risk, early detection, diagnosis, prognosis as well as to discuss the discovery of new targets for prevention and therapy. It is anticipated that such markers and targets will be based on cellular, subcellular, molecular and functional aberrations within the lesion and within individual cells. Additionally, single-cell proteomic data will be essential for the establishment of new tools that with searchable and scalable features that include spatial and temporal cartographies of premalignant and malignant lesions. Challenges and potential solutions that were discussed included: • The best way/s to analyze single-cells from fresh and preserved tissue • Detection and analysis of secreted molecules and from single cells, especially from a tissue slice • Detection of new, previously undocumented cell type/s in the premalignant and early stage cancer tissue microenvironment • Multi-omic integration of data to support and inform proteomic measurements • Subcellular organelles – identifying abnormal structure, function, distribution and location within individual premalignant and malignant cells • How to improve the dynamic range of single-cell proteomic measurements for discovery of differentially expressed proteins and their post translational modifications (PTM) • The depth of coverage measured concurrently using single-cell techniques • Quantitation - absolute or semiquantitative? • Single methodology or multiplexed combinations? • Application of analytical methods for identification of biologically significant subsets • Data visualization of N-dimensional datasets • How to construct intercellular signaling networks in individual cells within premalignant tumor microenvironments (TME) • Associations between intrinsic cellular processes and extrinsic stimuli • How to predict cellular responses to stress inducing stimuli • Identification of new markers for prediction of progression from precursor, benign and localized lesions to invasive cancer, based on spatial and temporal changes within individual cells • Identification of new targets for immunoprevention or immunotherapy – identification of neoantigens and surfactome of individual cells within a lesion

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1997

Determining divergence times with a protein clock: update and reevaluation

A recent study of the divergence times of the major groups of organisms as gauged by amino acid sequence comparison has been expanded and the data have been reanalyzed with a distance measure that corrects for both constraints on amino acid interchange and variation in substitution rate at different sites. Beyond that, the availability of complete genome sequences for several eubacteria and an archaebacterium has had a great impact on the interpretation of certain aspects of the data. Thus, the majority of the archaebacterial sequences are not consistent with currently accepted views of the Tree of Life which cluster the archaebacteria with eukaryotes. Instead, they are either outliers or mixed in with eubacterial orthologs. The simplest resolution of the problem is to postulate that many of these sequences were carried into eukaryotes by early eubacterial endosymbionts about 2 billion years ago, only very shortly after or even coincident with the divergence of eukaryotes and archaebacteria. The strong resemblances of these same enzymes among the major eubacterial groups suggest that the cyanobacteria and Gram-positive and Gram-negative eubacteria also diverged at about this same time, whereas the much greater differences between archaebacterial and eubacterial sequences indicate these two groups may have diverged between 3 and 4 billion years ago.

NASA Discipline Exobiology↗
NASA NTRS1996

Chance and necessity in the selection of nucleic acid catalysts

In Tom Stoppard's famous play [Rosencrantz and Guildenstern are Dead], the ill-fated heroes toss a coin 101 times. The first 100 times they do so the coin lands heads up. The chance of this happening is approximately 1 in 10(30), a sequence of events so rare that one might argue that it could only happen in such a delightful fiction. Similarly rare events, however, may underlie the origins of biological catalysis. What is the probability that an RNA, DNA, or protein molecule of a given random sequence will display a particular catalytic activity? The answer to this question determines whether a collection of such sequences, such as might result from prebiotic chemistry on the early earth, is extremely likely or unlikely to contain catalytically active molecules, and hence whether the origin of life itself is a virtually inevitable consequence of chemical laws or merely a bizarre fluke. The fact that a priori estimates of this probability, given by otherwise informed chemists and biologists, ranged from 10(-5) to 10(-50), inspired us to begin to address the question experimentally. As it turns out, the chance that a given random sequence RNA molecule will be able to catalyze an RNA polymerase-like phosphoryl transfer reaction is close to 1 in 10(13), rare enough, to be sure, but nevertheless in a range that is comfortably accessible by experiment. It is the purpose of this Account to describe the recent advances in combinatorial biochemistry that have made it possible for us to explore the abundance and diversity of catalysts existing in nucleic acid sequence space.

Review, Tutorial↗
DOE OSTI2021

A small molecule antagonizes jasmonic acid perception and auxin responses in vascular and nonvascular plants

The phytohormone jasmonoyl-L-isoleucine (JA-Ile) regulates many stress responses and developmental processes in plants. A co-receptor complex formed by the F-box protein Coronatine Insensitive 1 (COI1) and a Jasmonate (JA) ZIM-domain (JAZ) repressor perceives the hormone. JA-Ile antagonists are invaluable tools for exploring the role of JA-Ile in specific tissues and developmental stages, and for identifying regulatory processes of the signaling pathway. Using two complementary chemical screens, we identified three compounds that exhibit a robust inhibitory effect on both the hormone-mediated COI–JAZ interaction and degradation of JAZ1 and JAZ9 in vivo. One molecule, J4, also restrains specific JA-induced physiological responses in different angiosperm plants, including JA-mediated gene expression, growth inhibition, chlorophyll degradation, and anthocyanin accumulation. Interaction experiments with purified proteins indicate that J4 directly interferes with the formation of the Arabidopsis (Arabidopsis thaliana) COI1–JAZ complex otherwise induced by JA. The antagonistic effect of J4 on COI1–JAZ also occurs in the liverwort Marchantia polymorpha, suggesting the mode of action is conserved in land plants. Besides JA signaling, J4 works as an antagonist of the closely related auxin signaling pathway, preventing Transport Inhibitor Response1/Aux–indole-3-acetic acid interaction and auxin responses in planta, including hormone-mediated degradation of an auxin repressor, gene expression, and gravitropic response. However, J4 does not affect other hormonal pathways. Altogether, our results show that this dual antagonist competes with JA-Ile and auxin, preventing the formation of phylogenetically related receptor complexes. J4 may be a useful tool to dissect both the JA-Ile and auxin pathways in particular tissues and developmental stages since it reversibly inhibits these pathways. One-sentence summary: A chemical screen identified a molecule that antagonizes jasmonate perception by directly interfering with receptor complex formation in phylogenetically distant vascular and nonvascular plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Workflow for High-throughput Screening of Enzyme Mutant Libraries Using Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Analysis of Escherichia coli Colonies

High-throughput molecular screening of microbial colonies and DNA libraries are critical procedures that enable applications such as directed evolution, functional genomics, microbial identification, and creation of engineered microbial strains to produce high-value molecules. A promising chemical screening approach is the measurement of products directly from microbial colonies via optically guided matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Measuring the compounds from microbial colonies bypasses liquid culture with a screen that takes approximately 5 s per sample. We describe a protocol combining a dedicated informatics pipeline and sample preparation method that can prepare up to 3,000 colonies in under 3 h. The screening protocol starts from colonies grown on Petri dishes and then transferred onto MALDI plates via imprinting. The target plate with the colonies is imaged by a flatbed scanner and the colonies are located via custom software. The target plate is coated with MALDI matrix, MALDI-MS analyzes the colony locations, and data analysis enables the determination of colonies with the desired biochemical properties. This workflow screens thousands of colonies per day without requiring additional automation. The wide chemical coverage and the high sensitivity of MALDI-MS enable diverse screening projects such as modifying enzymes and functional genomics surveys of gene activation/inhibition libraries.

Choe, Kisurb↗
NASA NTRS2002

AmphiFoxE4, an amphioxus winged helix/forkhead gene encoding a protein closely related to vertebrate thyroid transcription factor-2: expression during pharyngeal development

The full-length sequence and developmental expression of amphioxus AmphiFoxE4 are described. Transcripts of the gene are first detected in the pharyngeal endoderm, where the club-shaped gland is forming and subsequently in the definitive gland itself. AmphiFoxE4 is closely related to vertebrate genes encoding the thyroid-specific transcription factor-2 (TTF2), which plays an early developmental role in the morphogenesis of the thyroid gland and a later role in hormone-mediated control of thyroid function. In amphioxus, AmphiFoxE4 expression is not thyroid specific because the club-shaped gland, the only structure expressing the gene, is not homologous to the vertebrate thyroid; instead, the thyroid homologue of amphioxus is a specialized region of the pharyngeal endoderm called the endostyle. We propose that (a) the pharynx of an amphioxus-like ancestor of the vertebrates included a club-shaped gland that expressed FoxE4 as well as an endostyle that did not, and (b) the club-shaped gland soon disappeared in the vertebrate line of descent but (c) not before there was a homeogenetic transfer of FoxE4 expression from the club-shaped gland to the nearby endostyle. Such a transfer could have provided part of the genetic program enabling the endostyle to separate from the pharyngeal endoderm and migrate away as the rudiment of the thyroid gland.

Non-NASA Center↗
DOE OSTI2023

Systematic and scalable genome-wide essentiality mapping to identify nonessential genes in phages

Phages are one of the key ecological drivers of microbial community dynamics, function, and evolution. Despite their importance in bacterial ecology and evolutionary processes, phage genes are poorly characterized, hampering their usage in a variety of biotechnological applications. Methods to characterize such genes, even those critical to the phage life cycle, are labor intensive and are generally phage specific. Here, we develop a systematic gene essentiality mapping method scalable to new phage–host combinations that facilitate the identification of nonessential genes. As a proof of concept, we use an arrayed genome-wide CRISPR interference (CRISPRi) assay to map gene essentiality landscape in the canonical coliphages λ and P1. Results from a single panel of CRISPRi probes largely recapitulate the essential gene roster determined from decades of genetic analysis for lambda and provide new insights into essential and nonessential loci in P1. We present evidence of how CRISPRi polarity can lead to false positive gene essentiality assignments and recommend caution towards interpreting CRISPRi data on gene essentiality when applied to less studied phages. Finally, we show that we can engineer phages by inserting DNA barcodes into newly identified inessential regions, which will empower processes of identification, quantification, and tracking of phages in diverse applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Binding of the substrate analog methanol in the oxygen-evolving complex of photosystem II in the D1-N87A genetic variant of cyanobacteria

The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive a catalyst capable of oxidizing water. The water oxidation reaction is catalyzed at the Mn 4 Ca-oxo cluster in the oxygen-evolving complex (OEC), which cycles through five light-driven S-state intermediates (S 0 –S 4 ). A detailed mechanism of the reaction remains elusive as it requires knowledge of the delivery and binding of substrate water in the higher S-state intermediates. In this paper, we use two-dimensional (2D) hyperfine sublevel correlation spectroscopy, in conjunction with quantum mechanics/molecular mechanics (QM/MM) and density functional theory (DFT), to probe the binding of the substrate analog, methanol, in the S 2 state of the D1-N87A variant of PSII from Synechocystis sp. PCC 6803. The results indicate that the size and specificity of the “narrow” channel is altered in D1-N87A PSII, allowing for the binding of deprotonated 13 C-labeled methanol at the Mn4(IV) ion of the catalytic cluster in the S 2 state. This has important implications on the mechanistic models for water oxidation in PSII.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2001

Leiomodins: larger members of the tropomodulin (Tmod) gene family

The 64-kDa autoantigen D1 or 1D, first identified as a potential autoantigen in Graves' disease, is similar to the tropomodulin (Tmod) family of actin filament pointed end-capping proteins. A novel gene with significant similarity to the 64-kDa human autoantigen D1 has been cloned from both humans and mice, and the genomic sequences of both genes have been identified. These genes form a subfamily closely related to the Tmods and are here named the Leiomodins (Lmods). Both Lmod genes display a conserved intron-exon structure, as do three Tmod genes, but the intron-exon structure of the Lmods and the Tmods is divergent. mRNA expression analysis indicates that the gene formerly known as the 64-kDa autoantigen D1 is most highly expressed in a variety of human tissues that contain smooth muscle, earning it the name smooth muscle Leiomodin (SM-Lmod; HGMW-approved symbol LMOD1). Transcripts encoding the novel Lmod gene are present exclusively in fetal and adult heart and adult skeletal muscle, and it is here named cardiac Leiomodin (C-Lmod; HGMW-approved symbol LMOD2). Human C-Lmod is located near the hypertrophic cardiomyopathy locus CMH6 on human chromosome 7q3, potentially implicating it in this disease. Our data demonstrate that the Lmods are evolutionarily related and display tissue-specific patterns of expression distinct from, but overlapping with, the expression of Tmod isoforms. Copyright 2001 Academic Press.

Carrier Proteins/biosynthesis/genetics↗
NASA NTRS2003

Conservation of Endo16 expression in sea urchins despite evolutionary divergence in both cis and trans-acting components of transcriptional regulation

Evolutionary changes in transcriptional regulation undoubtedly play an important role in creating morphological diversity. However, there is little information about the evolutionary dynamics of cis-regulatory sequences. This study examines the functional consequence of evolutionary changes in the Endo16 promoter of sea urchins. The Endo16 gene encodes a large extracellular protein that is expressed in the endoderm and may play a role in cell adhesion. Its promoter has been characterized in exceptional detail in the purple sea urchin, Strongylocentrotus purpuratus. We have characterized the structure and function of the Endo16 promoter from a second sea urchin species, Lytechinus variegatus. The Endo16 promoter sequences have evolved in a strongly mosaic manner since these species diverged approximately 35 million years ago: the most proximal region (module A) is conserved, but the remaining modules (B-G) are unalignable. Despite extensive divergence in promoter sequences, the pattern of Endo16 transcription is largely conserved during embryonic and larval development. Transient expression assays demonstrate that 2.2 kb of upstream sequence in either species is sufficient to drive GFP reporter expression that correctly mimics this pattern of Endo16 transcription. Reciprocal cross-species transient expression assays imply that changes have also evolved in the set of transcription factors that interact with the Endo16 promoter. Taken together, these results suggest that stabilizing selection on the transcriptional output may have operated to maintain a similar pattern of Endo16 expression in S. purpuratus and L. variegatus, despite dramatic divergence in promoter sequence and mechanisms of transcriptional regulation.

NASA Discipline Evolutionary Biology↗
DOE OSTI2023

High-Throughput Characterization and Optimization of Polyamide Hydrolase Activity Using Open Port Sampling Interface Mass Spectrometry

Enzymatic biodegradation of polymers, such as polyamides (PA), has the potential to cost-effectively reduce plastic waste, but enhancements in degradation efficiency are needed. Engineering enzymes through directed evolution is one pathway toward identification of critical domains needed for improving activity. However, screening such enzymatic libraries (100s-to-1000s of samples) is time-consuming. Here we demonstrate the use of robotic autosampler (PAL) and immediate drop on demand technology (I.DOT) liquid handling systems coupled with open-port sampling interface-mass spectrometry (OPSI-MS) to screen for PA6 and PA66 hydrolysis by 6-aminohexanoate-oligomer endo-hydrolase (nylon hydrolase, NylC) in a high-throughput (8–20 s/sample) manner. The OPSI-MS technique required minimal sample preparation and was amenable to 96-well plate formats for automated processing. Enzymatic hydrolysis of PA characteristically produced soluble linear oligomer products that could be identified by OPSI-MS. Incubation temperatures and times were optimized for PA6 (65 °C, 24 h) and PA66 (75 °C, 24 h) over 108 experiments. In addition, the I.DOT/OPSI-MS quantified production of PA6 linear dimer (8.3 ± 1.6 μg/mL) and PA66 linear monomer (13.5 ± 1.5 μg/mL) by NylC with a lower limit of detection of 0.029 and 0.032 μg/mL, respectively. For PA6 and PA66, linear oligomer production corresponded to 0.096 ± 0.018% and 0.204 ± 0.028% conversion of dry pellet mass, respectively. The developed methodology is expected to be utilized to assess enzymatic hydrolysis of engineered enzyme libraries, comprising hundreds to thousands of individual samples.

47 OTHER INSTRUMENTATION↗
DOE OSTI2025

Rewiring Aromatic Compound Consumption: Chromosomal Amplification and Evolution of a Foreign Pathway in Acinetobacter baylyi ADP1

Rational engineering strategies that seek to harness the remarkable diversity of microbial metabolism can be limited by incomplete biological knowledge. As described here, a novel approach to address this challenge involved replacing a native pathway for degrading lignin-derived aromatic compounds via ortho cleavage of protocatechuate in Acinetobacter baylyi ADP1 with a foreign meta-cleavage pathway that uses different enzymes, metabolites, and redox carriers. This alteration may improve lignin valorization and coordinate catabolism with bioproduction strategies. When a 14-kbp region of foreign DNA was inserted in the chromosome, the heterologous genes failed to confer growth on target substrates. Regional gene dosage was increased using a synthetic DNA fragment to promote recombination, and higher copy number enabled growth. During adaptive laboratory evolution, compensatory mutations arose that permit growth with one copy of the foreign genes. This complex metabolic remodeling was accomplished without assumptions about the impediments that initially prevented growth. To understand the changes that emerged, a novel transformation assay identified a combination of mutations sufficient for the new phenotype. Three unexpected changes were revealed: loss of one foreign enzyme, loss of one native enzyme, and loss of a two-component transcriptional regulatory system. This study establishes that large multicopy tandem arrays of poorly adapted pathway genes can confer new functions and improve understanding of metabolism.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗