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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 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Quantitative trait locus mapping combined with variant and transcriptome analyses identifies a cluster of gene candidates underlying the variation in leaf wax between upland and lowland switchgrass ecotypes

Switchgrass (Panicum virgatum L.) is a promising warm-season candidate energy crop. It occurs in two ecotypes, upland and lowland, which vary in a number of phenotypic traits, including leaf glaucousness. To initiate trait mapping, two F 2 mapping populations were developed by crossing two different F 1 sibs derived from a cross between the tetraploid lowland genotype AP13 and the tetraploid upland genotype VS16, and high-density linkage maps were generated. Quantitative trait locus (QTL) analyses of visually scored leaf glaucousness and of hydrophobicity of the abaxial leaf surface measured using a drop shape analyzer identified highly significant colocalizing QTL on chromosome 7K (Chr07K). Using a multipronged approach, we identified a cluster of genes including Pavir.7KG077009, which encodes a Type III polyketide synthase-like protein, and Pavir.7KG013754 and Pavir.7KG030500, two highly similar genes that encode putative acyl-acyl carrier protein (ACP) thioesterases, as strong candidates underlying the QTL. The lack of homoeologs for any of the three genes on Chr07N, the relatively low level of identity with other switchgrass KCS proteins and thioesterases, as well as the organization of the surrounding region suggest that Pavir.7KG077009 and Pavir.7KG013754/Pavir.7KG030500 were duplicated into a fast-evolving chromosome region, which led to their neofunctionalization. Furthermore, sequence analyses showed all three genes to be absent in the two upland compared to the two lowland accessions analyzed. This study provides an example of and practical guide for trait mapping and candidate gene identification in a complex genetic system by combining QTL mapping, transcriptomics and variant analysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Major involvement of two laccase genes in conidial pigment biosynthesis in Aspergillus oryzae

Wild-type strains of Aspergillus oryzae develop yellow, yellow-green, green, or brown conidia. Previous reports suggested that the conidiation initiates with the biosynthesis of a yellow pigment YWA1 from acetyl-CoA by a polyketide synthase encoded by wA (AO090102000545). This is followed by the conversion to other pigment by a laccase encoded by yA (AO090011000755). Based on orthologous pathways in other aspergilli, it is reasonable to hypothesize that in addition to yA, AO090102000546 encoding laccase and AO090005000332 encoding Ayg1-like hydrolase play a role in A. oryzae conidial pigment biosynthesis. However, the involvement of these two genes in conidial pigmentation remains unclear. In this study, we tested this hypothesis by assessing the conidial colors of both disruption and overexpression mutants to verify whether AO090102000546 and AO090005000332 were associated with the conidial pigmentation. Here, observation of single, double, and triple disruptants of these three genes suggested that conidial pigments were synthesized by two laccase genes, AO090011000755 and AO090102000546, whereas Ayg1-like hydrolase gene AO090005000332 was proven to have no obvious association with the synthesis. This was corroborated by observing the phenotype of each overexpression mutant. Interestingly, AO090005000332 overexpression mutant produced smoky yellow-green conidia, different from the wild-type strain. Thus, the AO090005000332-encoded protein is likely to maintain the enzymatic activity. However, the expression level was observed to be one-third of that of AO090102000546 and one-seventh of that of AO090011000755. Consequently, apparent lack of obvious contribution of AO090005000332 to conidial pigmentation could be attributed to its low expression level. Expression analysis indicated similar profiles in several wild-type strains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Expression of blue pigment synthetase a from Streptomyces lavenduale reveals insights on the effects of refactoring biosynthetic megasynthases for heterologous expression in Escherichia coli .

High GC bacteria from the genus Streptomyces harbor expansive secondary metabolism. The expression of biosynthetic proteins and the characterization and identification of biological "parts" for synthetic biology purposes from such pathways are of interest. However, the high GC content of proteins from actinomycetes in addition to the large size and multi-domain architecture of many biosynthetic proteins (such as non-ribosomal peptide synthetases; NRPSs, and polyketide synthases; PKSs often called "megasynthases") often presents issues with full-length translation and folding. Here we evaluate a non-ribosomal peptide synthetase (NRPS) from Streptomyces lavenduale, a multidomain "megasynthase" gene that comes from a high GC (72.5%) genome. While a preliminary step in revealing differences, to our knowledge this presents the first head-to-head comparison of codon-optimized sequences versus a native sequence of proteins of streptomycete origin heterologously expressed in E. coli. We found that any disruption in co-translational folding from codon mismatch that reduces the titer of indigoidine is explainable via the formation of more inclusion bodies as opposed to compromising folding or posttranslational modification in the soluble fraction. In conclusion, this result supports that one could apply any refactoring strategies that improve soluble expression in E. coli without concern that the protein that reaches the soluble fraction is differentially folded.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Hybrid biological-chemical strategy for converting polyethylene into a recyclable plastic monomer using engineered Corynebacterium glutamicum

Converting polyethylene (PE) into valuable materials, particularly ones that are better for the environment than the incumbent plastics, not only helps mitigate environmental issues caused by plastic waste but also alleviates the long-standing problem of microbial fermentation competing with food supplies. However, the inherent robustness of PE due to its strong carbon-carbon bonds and high molecular weight necessitates harsh decomposition conditions, resulting in diverse decomposition outcomes that present significant challenges for downstream applications, especially for bioconversion. In this study, we demonstrate a hybrid biological-chemical conversion process for PE, converting its decomposition products, namely short-chain diacids, into a monomer, β-keto-δ-lactone (BKDL), for highly recyclable polydiketoenimine plastics using engineered Corynebacterium glutamicum. Since BKDL synthesis requires a substantial supply of malonyl-CoA, we employed an alternative biosynthesis pathway that leverages C. glutamicum's natural proficiency in amino acid production. We optimized this pathway in vivo by minimizing carbon loss to CO2 and byproducts, improving the transporter system, and maximizing co-factor regeneration. Furthermore, we co-optimized the PE deconstruction process to produce predominantly C4 to C6 diacids and integrated three catabolic pathways into the engineered strain to enhance diacid utilization, maximizing the carbon conversion from PE. Finally, an engineered polyketide synthase was introduced into C. glutamicum to enable BKDL synthesis. This work demonstrates the potential of a chemo-biological hybrid strategy for recycling plastic waste, highlighting its promise in addressing environmental challenges and promoting sustainable materials.

Zhan, Chunjun↗
DOE OSTI2025

Functional and Structural Studies on the Esperamicin Thioesterase and Progress toward Understanding Enediyne Core Biosynthesis

Enediynes are among the most potent antitumor and antibacterial natural products. Studies on their biosynthetic pathways have identified a shared, linear polyene precursor generated from an iterative type I polyketide synthase (PKSE) as the source of the enediyne warhead. A key step is the release of this polyene from the PKSE by a discrete thioesterase (TE). Here, in this study, we used X-ray crystallography, site-directed mutagenesis, and heterologous coexpression of PKSEs and TEs to elucidate how enediyne TEs mediate the production of the polyene. We solved the structure of wild-type EspE7 from esperamicin producer Actinomodura verrucosospora. The substrate binding pocket was also defined upon serendipitous cocrystallization of an EspE7 mutant with a fatty acyl-CoA ligand. Structural data and in vitro activity assays with EspE7 mutants provide strong evidence that Glu68 in EspE7 and the analogous Glu residue in other enediyne TEs functions as a key catalytic residue, thus supporting a hydrolysis mechanism for enediyne TEs that aligns with that of Pseudomonas sp. 4-HB-CoA TE. Furthermore, combinations of 9- and 10-membered enediyne PKSEs and TEs produced 1,3,5,7,9,11,13-pentadecaheptaene (1) as the major product. Thus, the data further support previous conclusions that 1 serves as the sole precursor for the biosynthesis of all enediyne cores.

Enediynes↗
DOE OSTI2021

Synthase-Selective Exploration of a Tunicate Microbiome by Activity-Guided Single-Cell Genomics

While thousands of environmental metagenomes have been mined for the presence of novel biosynthetic gene clusters, such computational predictions do not provide evidence of their in vivo biosynthetic functionality. Using fluorescent in situ enzyme assay targeting carrier proteins common to polyketide (PKS) and nonribosomal peptide synthetases (NRPS), we applied fluorescence-activated cell sorting to tunicate microbiome to enrich for microbes with active secondary metabolic capabilities. Single-cell genomics uncovered the genetic basis for a wide biosynthetic diversity in the enzyme-active cells and revealed a member of marine Oceanospirillales harboring a novel NRPS gene cluster with high similarity to phylogenetically distant marine and terrestrial bacteria. Interestingly, this synthase belongs to a larger class of siderophore biosynthetic gene clusters commonly associated with pestilence and disease. Furthermore, this demonstrates activity-guided single-cell genomics as a tool to guide novel biosynthetic discovery.

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

Heterologous Expression of Cryptomaldamide in a Cyanobacterial Host

Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, nonribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drug leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that is typically available from the native organisms. Additionally, investigations of the biosynthetic gene clusters and enzymatic pathways have been difficult due to the inability to conduct genetic manipulations in the native producers. Here we report a set of genetic tools for the heterologous expression of biosynthetic gene clusters in the cyanobacteria Synechococcus elongatus PCC 7942 and Anabaena (Nostoc) PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of Anabaena that contains S. elongatus homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of Anabaena. These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium Moorena (Moorea) producens JHB in both model strains. S. elongatus did not produce cryptomaldamide; however, high-titer production of cryptomaldamide was obtained in Anabaena. Furthermore, the methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Seasonal activities of the phyllosphere microbiome of perennial crops

Understanding the interactions between plants and microorganisms can inform microbiome management to enhance crop productivity and resilience to stress. Here, we apply a genome-centric approach to identify ecologically important leaf microbiome members on replicated plots of field-grown switchgrass and miscanthus, and to quantify their activities over two growing seasons for switchgrass. We use metagenome and metatranscriptome sequencing and curate 40 medium- and high-quality metagenome-assembled-genomes (MAGs). We find that classes represented by these MAGs (Actinomycetia, Alpha- and Gamma- Proteobacteria, and Bacteroidota) are active in the late season, and upregulate transcripts for short-chain dehydrogenase, molybdopterin oxidoreductase, and polyketide cyclase. Stress-associated pathways are expressed for most MAGs, suggesting engagement with the host environment. We also detect seasonally activated biosynthetic pathways for terpenes and various non-ribosomal peptide pathways that are poorly annotated. Our findings support that leaf-associated bacterial populations are seasonally dynamic and responsive to host cues.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-$α$-amino acids in vitro and in vivo

The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-L-α-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Here we report that pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants accept α-hydroxy, α-thio and N-formyl-L-α-amino acids, as well as α-carboxy acid monomers that are precursors to polyketide natural products. These monomers are accommodated and accepted by the translation apparatus in vitro; those with reactive nucleophiles are incorporated into proteins in vivo. High-resolution structural analysis of the complex formed between one PylRS enzyme and a m-substituted 2-benzylmalonic acid derivative revealed an active site that discriminates prochiral carboxylates and accommodates the large size and distinct electrostatics of an α-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges substantially from the α-amine of proteinogenic amino acids. These enzymes or derivatives thereof could synergize with natural or evolved ribosomes and/or translation factors to generate diverse sequence-defined non-protein heteropolymers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Complete biosynthesis of QS-21 in engineered yeast

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host’s native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families—a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases—from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure–activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Comparative and pangenomic analysis of the genus Streptomyces

Abstract Streptomycetes are highly metabolically gifted bacteria with the abilities to produce bioproducts that have profound economic and societal importance. These bioproducts are produced by metabolic pathways including those for the biosynthesis of secondary metabolites and catabolism of plant biomass constituents. Advancements in genome sequencing technologies have revealed a wealth of untapped metabolic potential from Streptomyces genomes. Here, we report the largest Streptomyces pangenome generated by using 205 complete genomes. Metabolic potentials of the pangenome and individual genomes were analyzed, revealing degrees of conservation of individual metabolic pathways and strains potentially suitable for metabolic engineering. Of them, Streptomyces bingchenggensis was identified as a potent degrader of plant biomass. Polyketide, non-ribosomal peptide, and gamma-butyrolactone biosynthetic enzymes are primarily strain specific while ectoine and some terpene biosynthetic pathways are highly conserved. A large number of transcription factors associated with secondary metabolism are strain-specific while those controlling basic biological processes are highly conserved. Although the majority of genes involved in morphological development are highly conserved, there are strain-specific varieties which may contribute to fine tuning the timing of cellular differentiation. Overall, these results provide insights into the metabolic potential, regulation and physiology of streptomycetes, which will facilitate further exploitation of these important bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Biorenewable and circular polydiketoenamine plastics

Amid growing concerns over the human health and environmental impacts of plastic waste, the most promising solution would be to build a circular plastics economy where sustainability considerations dictate the full life cycle of plastics use including replacing petrochemicals with biorenewables. Here we show that by incorporating the polyketide triacetic acid lactone (TAL) in polydiketoenamines (PDK) we increase the working temperature of these circular plastics, opening the door wider to applications where circularity is urgently needed. By varying the number of carbons of TAL-derived monomers, both polymer properties and recycling efciency are afected. Simply using glucose as the main carbon source, we engineered a process for producing bioTAL under fed-batch fermentation. Furthermore, a systems analysis of this bioprocess under diferent scenarios quantifes the environmental and economic benefts of PDK plastics and the risks when implemented at an industrial scale, providing opportunities in biorenewable circularity.

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

Synergism of anisotropic and computational NMR methods reveals the likely configuration of phormidolide A

Characterization of the complex molecular scaffold of the marine polyketide natural product phormidolide A represents a challenge that has persisted for nearly two decades. In light of discordant results arising from recent synthetic and biosynthetic reports, a rigorous study of the configuration of phormidolide A was necessary. This report outlines a synergistic effort employing computational and anisotropic NMR investigation, that provided orthogonal confirmation of the reassigned side chain, as well as supporting a further correction of the C7 stereocenter.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Cell-free synthetic biology for natural product biosynthesis and discovery

Natural products have applications as biopharmaceuticals, agrochemicals, and other high-value chemicals. However, there are challenges in isolating natural products from their native producers (e.g. bacteria, fungi, plants). In many cases, synthetic chemistry or heterologous expression must be used to access these important molecules. The biosynthetic machinery to generate these compounds is found within biosynthetic gene clusters, primarily consisting of the enzymes that biosynthesise a range of natural product classes (including, but not limited to ribosomal and nonribosomal peptides, polyketides, and terpenoids). Cell-free synthetic biology has emerged in recent years as a bottom-up technology applied towards both prototyping pathways and producing molecules. Recently, it has been applied to natural products, both to characterise biosynthetic pathways and produce new metabolites. This review discusses the core biochemistry of cell-free synthetic biology applied to metabolite production and critiques its advantages and disadvantages compared to whole cell and/or chemical production routes. Specifically, we review the advances in cell-free biosynthesis of ribosomal peptides, analyse the rapid prototyping of natural product biosynthetic enzymes and pathways, highlight advances in novel antimicrobial discovery, and discuss the rising use of cell-free technologies in industrial biotechnology and synthetic biology.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Engineering modular enzyme assembly: synthetic interface strategies for natural products biosynthesis applications

Covering: 2020 to 2025Natural products remain indispensable sources of therapeutic and bioactive compounds, yet traditional discovery strategies are constrained by compound rediscovery. Modular biosynthetic enzymes, such as type I polyketide synthases (PKSs) and type A non-ribosomal peptide synthetases (NRPSs), offer promising platforms for combinatorial biosynthesis owing to their programmable architectures. However, practical implementation is frequently limited by inter-modular incompatibility and domain-specific interactions. This review highlights recent advances in modular enzyme assembly enabled by synthetic interfaces-including cognate docking domains, synthetic coiled-coils, SpyTag/SpyCatcher, and split inteins-which function as orthogonal, standardized connectors to facilitate post-translational complex formation. These interfaces support rational investigations into substrate specificity, module compatibility, and pathway derivatization as well as general enzyme clustering applications beyond PKS and NRPS systems. Synthetic interfaces can be integrated with computational tools to support a more systematic and scalable framework for modular enzyme engineering by providing predictive insights into domain compatibility and interface design. These approaches within iterative design-build-test-learn workflows can accelerate the programmable assembly of biosynthetic systems and expand the accessible chemical space for natural products.

Kim, Gahyeon↗
DOE OSTI2021

Anaerobic gut fungi are an untapped reservoir of natural products

Anaerobic fungi (class Neocallimastigomycetes) thrive as low-abundance members of the herbivore digestive tract. The genomes of anaerobic gut fungi are poorly characterized and have not been extensively mined for the biosynthetic enzymes of natural products such as antibiotics. Here, we investigate the potential of anaerobic gut fungi to synthesize natural products that could regulate membership within the gut microbiome. Here, complementary 'omics' approaches were combined to catalog the natural products of anaerobic gut fungi from four different representative species: Anaeromyces robustus (A. robustus), Caecomyces churrovis (C. churrovis), Neocallimastix californiae (N. californiae), and Piromyces finnis (P. finnis). In total, 146 genes were identified that encode biosynthetic enzymes for diverse types of natural products, including nonribosomal peptide synthetases and polyketide synthases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Reverse β-oxidation pathways for efficient chemical production

Abstract Microbial production of fuels, chemicals, and materials has the potential to reduce greenhouse gas emissions and contribute to a sustainable bioeconomy. While synthetic biology allows readjusting of native metabolic pathways for the synthesis of desired products, often these native pathways do not support maximum efficiency and are affected by complex regulatory mechanisms. A synthetic or engineered pathway that allows modular synthesis of versatile bioproducts with minimal enzyme requirement and regulation while achieving high carbon and energy efficiency could be an alternative solution to address these issues. The reverse β-oxidation (rBOX) pathways enable iterative non-decarboxylative elongation of carbon molecules of varying chain lengths and functional groups with only four core enzymes and no ATP requirement. Here, we describe recent developments in rBOX pathway engineering to produce alcohols and carboxylic acids with diverse functional groups, along with other commercially important molecules such as polyketides. We discuss the application of rBOX beyond the pathway itself by its interfacing with various carbon-utilization pathways and deployment in different organisms, which allows feedstock diversification from sugars to glycerol, carbon dioxide, methane, and other substrates.

59 BASIC BIOLOGICAL SCIENCES↗