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

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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Disruption of acyl-acyl carrier protein (acyl-ACP) synthetase in cyanobacteria impairs lipid remodeling as revealed by acyl-ACP measurements

Free fatty acid (FFA) production in bacteria is a key target for metabolic engineering. The knockout of the acyl-ACP synthetase (AAS) prevents reincorporation of FFA into the fatty acid biosynthetic cycle and is widely used to enhance their secretion. However, the role of AAS in membrane lipid remodeling under environmental stress, such as altered temperature, remains poorly understood. In cyanobacteria, temperature shifts are known to affect fatty acid desaturation and membrane fluidity, yet it is unclear whether AAS contributes to these adaptive responses through re-esterification of membrane-released acyl chains. We elucidated unique aspects of fatty acid metabolism in response to temperature changes in biotechnologically relevant microbes with the development of an efficient method for quantifying acyl-ACP intermediates using anion exchange chromatography (AEX). In Escherichia coli, which performs desaturation during fatty acid biosynthesis, we detected saturated and unsaturated acyl-ACPs that confirm biosynthetic pathway operation. In the cyanobacteria, Picosynechococcus sp. PCC 7002 and the Δaas strain, changes between two temperatures were interpreted with support from proteomic and lipidomic analyses and indicated that the AAS is tied to membrane lipid remodeling. Further, polyunsaturated acyl-ACPs were detected in the Δaas strain, which was unexpected because fatty acid synthesis does not produce polyunsaturates in cyanobacteria, suggesting the presence of alternative acyl-activating enzymes or unknown acyl-ACP desaturases. This study highlights the possible link between acyl chain recycling and lipid remodeling in cyanobacteria and demonstrates the utility of AEX-based acyl-ACP profiling in dissecting fatty acid metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. Furthermore, by activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein–protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Multiplexed CRISPR/Cas9 editing of the long-chain acyl-CoA synthetase family in the diatom Phaeodactylum tricornutum reveals that mitochondrial ptACSL3 is involved in the synthesis of storage lipids

Long-chain acyl-CoA synthetases (LACS) play diverse and fundamentally important roles in lipid metabolism. While their functions have been well established in bacteria, yeast and plants, the mechanisms by which LACS isozymes regulate lipid metabolism in unicellular oil-producing microalgae, including the diatom Phaeodactylum tricornutum , remain largely unknown. In P. tricornutum , a family of five genes ( ptACSL1 – ptACSL5 ) encodes LACS activities. For this work, we generated single lacs knockout/knockdown mutants using multiplexed CRISPR/Cas9 method, and determined their substrate specificities towards different fatty acids (FAs) and subcellular localisations. ptACSL3 is localised in the mitochondria and its disruption led to compromised growth and reduced triacylglycerol (TAG) content when cells were bubbled with air. The ptACSL3 mutants showed altered FA profiles in two galactoglycerolipids and phosphatidylcholine (PC) with significantly reduced distribution of 16:0 and 16:1. ptACSL5 is localised in the peroxisome and its knockdown resulted in reduced growth rate and altered molecular species of PC and TAG, indicating a role in controlling the composition of acyl-CoAs for lipid synthesis. Our work demonstrates the potential of generating gene knockout mutants with the mutation of large fragment deletion using multiplexed CRISPR/Cas9 and provides insight into the functions of LACS isozymes in lipid metabolism in the oleaginous microalgae.

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

Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module

Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and additional proteins that introduce chemical modifications before, during or after assembly-line synthesis. The bacillamide biosynthetic pathway is a common, three-protein system, with a decarboxylase that prepares an NRPS substrate, an NRPS, and an oxidase. Here, the pathway is reconstituted in vitro. The oxidase is shown to perform dehydrogenation of the thiazoline in the peptide intermediate while it is covalently attached to the NRPS, as the penultimate step in bacillamide D synthesis. Structural analysis of the oxidase reveals a dimeric, two-lobed architecture with a remnant RiPP recognition element and a dramatic wrapping loop. The oxidase forms a stable complex with the NRPS and dimerizes it. We visualized co-complexes of the oxidase bound to the elongation module of the NRPS using X-ray crystallography and cryo-EM. The three active sites (for adenylation, condensation/cyclization, and oxidation) form an elegant arc to facilitate substrate delivery. The structures enabled a proof-of-principle bioengineering experiment in which the BmdC oxidase domain is embedded into the NRPS.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A cryptic third active site in cyanophycin synthetase creates primers for polymerization

Cyanophycin is a nitrogen reserve biopolymer in many bacteria that has promising industrial applications. Made by cyanophycin synthetase 1 (CphA1), it has a poly-L-Asp backbone with L-Arg residues attached to each aspartate sidechain. CphA1s are thought to typically require existing segments of cyanophycin to act as primers for cyanophycin polymerization. In this study, we show that most CphA1s will not require exogenous primers and discover the surprising cause of primer independence: CphA1 can make minute quantities of cyanophycin without primer, and an unexpected, cryptic metallopeptidase-like active site in the N-terminal domain of many CphA1s digests these into primers, solving the problem of primer availability. We present co-complex cryo-EM structures, make mutations that transition CphA1s between primer dependence and independence, and demonstrate that primer dependence can be a limiting factor for cyanophycin production in heterologous hosts. In CphA1, domains with opposite catalytic activities combine into a remarkable, self-sufficient, biosynthetic nanomachine.

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

Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria

How bacteria sense and respond to nitrogen levels are central questions in microbial physiology. In Gram-positive bacteria, nitrogen homeostasis is controlled by an operon encoding glutamine synthetase (GS), a dodecameric machine that assimilates ammonium into glutamine, and the GlnR repressor. GlnR detects nitrogen excess indirectly by binding glutamine-feedback-inhibited-GS (FBI-GS), which activates its transcription-repression function. The molecular mechanisms behind this regulatory circuitry, however, are unknown. Here we describe biochemical and structural analyses of GS and FBI-GS-GlnR complexes from pathogenic and non-pathogenic Gram-positive bacteria. The structures show FBI-GS binds the GlnR C-terminal domain within its active-site cavity, juxtaposing two GlnR monomers to form a DNA-binding-competent GlnR dimer. The FBI-GS-GlnR interaction stabilizes the inactive GS conformation. Strikingly, this interaction also favors a remarkable dodecamer to tetradecamer transition in some GS, breaking the paradigm that all bacterial GS are dodecamers. These data thus unveil unique structural mechanisms of transcription and enzymatic regulation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Elucidating the molecular programming of a nonlinear non-ribosomal peptide synthetase responsible for fungal siderophore biosynthesis

Abstract Siderophores belonging to the ferrichrome family are essential for the viability of fungal species and play a key role for virulence of numerous pathogenic fungi. Despite their biological significance, our understanding of how these iron-chelating cyclic hexapeptides are assembled by non-ribosomal peptide synthetase (NRPS) enzymes remains poorly understood, primarily due to the nonlinearity exhibited by the domain architecture. Herein, we report the biochemical characterization of the SidC NRPS, responsible for construction of the intracellular siderophore ferricrocin. In vitro reconstitution of purified SidC reveals its ability to produce ferricrocin and its structural variant, ferrichrome. Application of intact protein mass spectrometry uncovers several non-canonical events during peptidyl siderophore biosynthesis, including inter-modular loading of amino acid substrates and an adenylation domain capable of poly-amide bond formation. This work expands the scope of NRPS programming, allows biosynthetic assignment of ferrichrome NRPSs, and sets the stage for reprogramming towards novel hydroxamate scaffolds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Phosphoribosylpyrophosphate synthetase as a metabolic valve advances Methylobacterium/Methylorubrum phyllosphere colonization and plant growth

The proficiency of phyllosphere microbiomes in efficiently utilizing plant-provided nutrients is pivotal for their successful colonization of plants. The methylotrophic capabilities of Methylobacterium/Methylorubrum play a crucial role in this process. However, the precise mechanisms facilitating efficient colonization remain elusive. In the present study, we investigate the significance of methanol assimilation in shaping the success of mutualistic relationships between methylotrophs and plants. A set of strains originating from Methylorubrum extorquens AM1 are subjected to evolutionary pressures to thrive under low methanol conditions. A mutation in the phosphoribosylpyrophosphate synthetase gene is identified, which converts it into a metabolic valve. This valve redirects limited C1-carbon resources towards the synthesis of biomass by up-regulating a non-essential phosphoketolase pathway. These newly acquired bacterial traits demonstrate superior colonization capabilities, even at low abundance, leading to increased growth of inoculated plants. This function is prevalent in Methylobacterium/Methylorubrum strains. In summary, our findings offer insights that could guide the selection of Methylobacterium/Methylorubrum strains for advantageous agricultural applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

ClusterCAD 2.0: an updated computational platform for chimeric type I polyketide synthase and nonribosomal peptide synthetase design

Abstract Megasynthase enzymes such as type I modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) play a central role in microbial chemical warfare because they can evolve rapidly by shuffling parts (catalytic domains) to produce novel chemicals. If we can understand the design rules to reshuffle these parts, PKSs and NRPSs will provide a systematic and modular way to synthesize millions of molecules including pharmaceuticals, biomaterials, and biofuels. However, PKS and NRPS engineering remains difficult due to a limited understanding of the determinants of PKS and NRPS fold and function. We developed ClusterCAD to streamline and simplify the process of designing and testing engineered PKS variants. Here, we present the highly improved ClusterCAD 2.0 release, available at https://clustercad.jbei.org. ClusterCAD 2.0 boasts support for PKS-NRPS hybrid and NRPS clusters in addition to PKS clusters; a vastly enlarged database of curated PKS, PKS-NRPS hybrid, and NRPS clusters; a diverse set of chemical ‘starters’ and loading modules; the new Domain Architecture Cluster Search Tool; and an offline Jupyter Notebook workspace, among other improvements. Together these features massively expand the chemical space that can be accessed by enzymes engineered with ClusterCAD.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The Structure and Immune Regulatory Implications of the Ubiquitin-Like Tandem Domain Within an Avian 2’-5’ Oligoadenylate Synthetase-Like Protein

Post-translational modification of host and viral proteins by ubiquitin and ubiquitin-like proteins plays a key role in a host’s ability to mount an effective immune response. Avian species lack a ubiquitin-like protein found in mammals and other non-avian reptiles; interferon stimulated gene product 15 (ISG15). ISG15 serves as a messenger molecule and can be conjugated to both host and viral proteins leading them to be stabilized, degraded, or sequestered. Structurally, ISG15 is comprised of a tandem ubiquitin-like domain (Ubl), which serves as the motif for post-translational modification. The 2’-5’ oligoadenylate synthetase-like proteins (OASL) also encode two Ubl domains in series near its C-terminus which binds OASL to retinoic acid inducible gene-I (RIG-I). This protein-protein interaction increases the sensitivity of RIG-I and results in an enhanced production of type 1 interferons and a robust immune response. Unlike human and other mammalian OASL homologues, avian OASLs terminate their tandem Ubl domains with the same LRLRGG motif found in ubiquitin and ISG15, a motif required for their conjugation to proteins. Chickens, however, lack RIG-I, raising the question of structural and functional characteristics of chicken OASL (chOASL). By investigating chOASL, the evolutionary history of viruses with deubiquitinases can be explored and drivers of species specificity for these viruses may be uncovered. Here we show that the chOASL tandem Ubl domains shares structural characteristics with mammalian ISG15, and that chOASL can oligomerize and conjugate to itself. In addition, the ISG15-like features of avian OASLs and how they impact interactions with viral deubiquitinases and deISGylases are explored.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1990

Glutamine synthetase immunor present in oligodendroglia of regions of the central nervous system

Glutamine synthetase immunoreactive oligodendrocytes were identified in the cerebral cortex, cerebellum, brain stem, and spinal cord. They were mostly confined to the gray matter, particularly close to neurons and processes. The white matter showed few immunoreactive oligodendroglia. It was suggested that some type of oligodendrocytes, specially those in perineuronal location, might fulfill a functional role more akin to astrocytes than to the normally myelinating oligodendroglia.

D'Amelio, Fernando↗