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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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All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Plant terpene specialized metabolism: complex networks or simple linear pathways?

From the perspectives of pathway evolution, discovery and engineering of plant specialized metabolism, the nature of the biosynthetic routes represents a critical aspect. Classical models depict biosynthesis typically from an end-point angle and as linear, for example, connecting central and specialized metabolism. As the number of functionally elucidated routes increased, the enzymatic foundation of complex plant chemistries became increasingly well understood. The perception of linear pathway models has been severely challenged. With a focus on plant terpenoid specialized metabolism, we review here illustrative examples supporting that plants have evolved complex networks driving chemical diversification. The completion of several diterpene, sesquiterpene and monoterpene routes shows complex formation of scaffolds and their subsequent functionalization. These networks show that branch points, including multiple sub-routes, mean that metabolic grids are the rule rather than the exception. This concept presents significant implications for biotechnological production.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Proteomic and metabolic disturbances in lignin-modified Brachypodium distachyon

Abstract Lignin biosynthesis begins with the deamination of phenylalanine and tyrosine (Tyr) as a key branch point between primary and secondary metabolism in land plants. Here, we used a systems biology approach to investigate the global metabolic responses to lignin pathway perturbations in the model grass Brachypodium distachyon. We identified the lignin biosynthetic protein families and found that ammonia-lyases (ALs) are among the most abundant proteins in lignifying tissues in grasses. Integrated metabolomic and proteomic data support a link between lignin biosynthesis and primary metabolism mediated by the ammonia released from ALs that is recycled for the synthesis of amino acids via glutamine. RNA interference knockdown of lignin genes confirmed that the route of the canonical pathway using shikimate ester intermediates is not essential for lignin formation in Brachypodium, and there is an alternative pathway from Tyr via sinapic acid for the synthesis of syringyl lignin involving yet uncharacterized enzymatic steps. Our findings support a model in which plant ALs play a central role in coordinating the allocation of carbon for lignin synthesis and the nitrogen available for plant growth. Collectively, these data also emphasize the value of integrative multiomic analyses to advance our understanding of plant metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Taxonomic and environmental distribution of bacterial amino acid auxotrophies

Abstract Many microorganisms are auxotrophic—unable to synthesize the compounds they require for growth. With this work, we quantify the prevalence of amino acid auxotrophies across a broad diversity of bacteria and habitats. We predicted the amino acid biosynthetic capabilities of 26,277 unique bacterial genomes spanning 12 phyla using a metabolic pathway model validated with empirical data. Amino acid auxotrophy is widespread across bacterial phyla, but we conservatively estimate that the majority of taxa (78.4%) are able to synthesize all amino acids. Our estimates indicate that amino acid auxotrophies are more prevalent among obligate intracellular parasites and in free-living taxa with genomic attributes characteristic of ‘streamlined’ life history strategies. We predicted the amino acid biosynthetic capabilities of bacterial communities found in 12 unique habitats to investigate environmental associations with auxotrophy, using data compiled from 3813 samples spanning major aquatic, terrestrial, and engineered environments. Auxotrophic taxa were more abundant in host-associated environments (including the human oral cavity and gut) and in fermented food products, with auxotrophic taxa being relatively rare in soil and aquatic systems. Overall, this work contributes to a more complete understanding of amino acid auxotrophy across the bacterial tree of life and the ecological contexts in which auxotrophy can be a successful strategy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Exploring single-cell biosynthetic noise and dynamics for enhanced betaxanthin production in Escherichia coli

Cell-to-cell variability often limits the efficiency of microbial bioproduction, yet how individual cells fluctuate over time and how these fluctuations shape population-level output remain unclear. To address this issue, we tracked a heterologous betaxanthin pathway in Escherichia coli using microfluidics-assisted time-lapse microscopy, allowing simultaneous measurement of fluctuations in betaxanthin, its biosynthetic enzyme DOD and growth across generations. Here we show that over 50% of high betaxanthin producers become medium or low producers after two divisions. Betaxanthin variation primarily originates from DOD noise, with a smaller contribution from growth rate fluctuations. We further develop a stochastic model to explore various control circuits and find that pathway enzyme or metabolite-based growth selection strategies are most effective in enhancing production. We experimentally validate the model by coupling enzyme expression to nutrient availability, which enriches high producers and boosts titer by 4.4-fold. Our results highlight key sources of metabolic heterogeneity and provide a framework for designing robust microbial processes.

Bacterial systems biology↗
DOE OSTI2023

Uncovering a miltiradiene biosynthetic gene cluster in the Lamiaceae reveals a dynamic evolutionary trajectory

The spatial organization of genes within plant genomes can drive evolution of specialized metabolic pathways. Terpenoids are important specialized metabolites in plants with diverse adaptive functions that enable environmental interactions. Here, we report the genome assemblies of Prunella vulgaris, Plectranthus barbatus, and Leonotis leonurus. We investigate the origin and subsequent evolution of a diterpenoid biosynthetic gene cluster (BGC) together with other seven species within the Lamiaceae (mint) family. Based on core genes found in the BGCs of all species examined across the Lamiaceae, we predict a simplified version of this cluster evolved in an early Lamiaceae ancestor. The current composition of the extant BGCs highlights the dynamic nature of its evolution. We elucidate the terpene backbones generated by the Callicarpa americana BGC enzymes, including miltiradiene and the terpene (+)-kaurene, and show oxidization activities of BGC cytochrome P450s. Our work reveals the fluid nature of BGC assembly and the importance of genome structure in contributing to the origin of metabolites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A symbiotic bacterium of Antarctic fish reveals environmental adaptability mechanisms and biosynthetic potential towards antibacterial and cytotoxic activities

Antarctic microbes are important agents for evolutionary adaptation and natural resource of bioactive compounds, harboring the particular metabolic pathways to biosynthesize natural products. However, not much is known on symbiotic microbiomes of fish in the Antarctic zone. In the present study, the culture method and whole-genome sequencing were performed. Natural product analyses were carried out to determine the biosynthetic potential. We report the isolation and identification of a symbiotic bacterium Serratia myotis L7-1, that is highly adaptive and resides within Antarctic fish, Trematomus bernacchii . As revealed by genomic analyses, Antarctic strain S. myotis L7-1 possesses carbohydrate-active enzymes (CAZymes), biosynthetic gene clusters (BGCs), stress response genes, antibiotic resistant genes (ARGs), and a complete type IV secretion system which could facilitate competition and colonization in the extreme Antarctic environment. The identification of microbiome gene clusters indicates the biosynthetic potential of bioactive compounds. Based on bioactivity-guided fractionation, serranticin was purified and identified as the bioactive compound, showing significant antibacterial and antitumor activity. The serranticin gene cluster was identified and located on the chrome. Furthermore, the multidrug resistance and strong bacterial antagonism contribute competitive advantages in ecological niches. Our results highlight the existence of a symbiotic bacterium in Antarctic fish largely represented by bioactive natural products and the adaptability to survive in the fish living in Antarctic oceans.

Xiao, Yu↗
DOE OSTI2022

Transcript and metabolite network perturbations in lignin biosynthetic mutants of Arabidopsis

Abstract Lignin, one of the most abundant polymers in plants, is derived from the phenylpropanoid pathway, which also gives rise to an array of metabolites that are essential for plant fitness. Genetic engineering of lignification can cause drastic changes in transcription and metabolite accumulation with or without an accompanying development phenotype. To understand the impact of lignin perturbation, we analyzed transcriptome and metabolite data from the rapidly lignifying stem tissue in 13 selected phenylpropanoid mutants and wild-type Arabidopsis (Arabidopsis thaliana). Our dataset contains 20,974 expressed genes, of which over 26% had altered transcript levels in at least one mutant, and 18 targeted metabolites, all of which displayed altered accumulation in at least one mutant. We found that lignin biosynthesis and phenylalanine supply via the shikimate pathway are tightly co-regulated at the transcriptional level. The hierarchical clustering analysis of differentially expressed genes (DEGs) grouped the 13 mutants into 5 subgroups with similar profiles of mis-regulated genes. Functional analysis of the DEGs in these mutants and correlation between gene expression and metabolite accumulation revealed system-wide effects on transcripts involved in multiple biological processes.

Plant Sciences↗
DOE OSTI2020

Comparative Genomic Analysis of Ochratoxin A Biosynthetic Cluster in Producing Fungi: New Evidence of a Cyclase Gene Involvement

The widespread use of Next-Generation Sequencing has opened a new era in the study of biological systems by significantly increasing the catalog of fungal genomes sequences and identifying gene clusters for known secondary metabolites as well as novel cryptic ones. However, most of these clusters still need to be examined in detail to completely understand the pathway steps and the regulation of the biosynthesis of metabolites. Genome sequencing approach led to the identification of the biosynthetic genes cluster of ochratoxin A (OTA) in a number of producing fungal species. Ochratoxin A is a potent pentaketide nephrotoxin produced by Aspergillus and Penicillium species and found as widely contaminant in food, beverages and feed. The increasing availability of several new genome sequences of OTA producer species in JGI Mycocosm and/or GenBank databanks led us to analyze and update the gene cluster structure in 19 Aspergillus and 2 Penicillium OTA producing species, resulting in a well conserved organization of OTA core genes among the species. Furthermore, our comparative genome analyses evidenced the presence of an additional gene, previously undescribed, located between the polyketide and non-ribosomal synthase genes in the cluster of all the species analyzed. The presence of a SnoaL cyclase domain in the sequence of this gene supports its putative role in the polyketide cyclization reaction during the initial steps of the OTA biosynthesis pathway. The phylogenetic analysis showed a clustering of OTA SnoaL domains in accordance with the phylogeny of OTA producing species at species and section levels. The characterization of this new OTA gene, its putative role and its expression evidence in three important representative producing species, are reported here for the first time.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Polyyne production is regulated by the transcriptional regulators PgnC and GacA in Pseudomonas protegens Pf-5

ABSTRACT Polyynes produced by bacteria have promising applications in agriculture and medicine due to their potent antimicrobial activities. Polyyne biosynthetic genes have been identified inPseudomonasandBurkholderia. However, the molecular mechanisms underlying the regulation of polyyne biosynthesis remain largely unknown. In this study, we used a soil bacteriumPseudomonas protegensPf-5, which was recently reported to produce polyyne called protegenin, as a model to investigate the regulation of bacterial polyyne production. Our results show that Pf-5 controls polyyne production at both the pathway-specific level and a higher global level. Mutation ofpgnC, a transcriptional regulatory gene located in the polyyne biosynthetic gene cluster, abolished polyyne production. Gene expression analysis revealed that PgnC directly activates the promoter of polyyne biosynthetic genes. The production of polyyne also requires a global regulator GacA. Mutation ofgacAdecreased the translation of PgnC, which is consistent with the result thatpgnCleader mRNA bound directly to RsmE, an RNA-binding protein negatively regulated by GacA. These results suggest that GacA induces the expression of the PgnC regulator, which in turn activates polyyne biosynthesis. Additionally, the polyyne-producing strain of Pf-5, but not the polyyne-nonproducing strain, could inhibit a broad spectrum of bacteria including both Gram-negative and Gram-positive bacteria. IMPORTANCE Antimicrobial metabolites produced by bacteria are widely used in agriculture and medicine to control plant, animal, and human pathogens. Although bacteria-derived polyynes have been identified as potent antimicrobials for decades, the molecular mechanisms by which bacteria regulate polyyne biosynthesis remain understudied. In this study, we found that polyyne biosynthesis is directly activated by a pathway-specific regulator PgnC, which is induced by a global regulator GacA through the RNA-binding protein RsmE inPseudomonas protegens. To our knowledge, this work is the first comprehensive study of the regulatory mechanisms of bacterial polyyne biosynthesis at both pathway-specific level and global level. The discovered molecular mechanisms can help us optimize polyyne production for agricultural or medical applications.

Biotechnology & Applied Microbiology↗
DOE OSTI2025

Discovery of Signaling Small Molecules (e.g. quorum sensing molecules) from the Microbiome

Microbial communities are shaped through the interactions between their microbial members and the environment (microbe-microbe and host-microbe interactions). Signal transduction pathways in the microbiome are often modulated through the small molecule products of microbial biosynthetic gene clusters (BGCs). Advances in 16S rRNA profiling and shotgun metagenomics have revolutionized our understanding about the microbial composition of various communities and their BGCs. Environmental metagenomes contain thousands of BGCs with uncharacterized small molecule products that potentially play roles in signal transduction. The overarching aim of this proposal was to develop computational techniques for discovering these small molecules and characterizing their bioactivity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Separate, separated, and together: the transcriptional program of the Clostridium acetobutylicum-Clostridium ljungdahlii syntrophy leading to interspecies cell fusion

ABSTRACT Syntrophic cocultures (hitherto assumed to be commensalistic) of Clostridium acetobutylicum and Clostridium ljungdahlii , whereby CO 2 and H 2 produced by the former feed the latter, result in interspecies cell fusion involving large-scale exchange of protein, RNA, and DNA between the two organisms. Although mammalian cell fusion is mechanistically dissected, the mechanism for such microbial-cell fusions is unknown. To start exploring this mechanism, we used RNA sequencing to identify genes differentially expressed in this coculture using two types of comparisons. One type compared coculture to the two monocultures, capturing the combined impact of interactions through soluble signals in the medium and through direct cell-to-cell interactions. The second type compared membrane-separated versus -unseparated cocultures, isolating the impact of interspecies physical contact. While we could not firmly identify specific genes that might drive cell fusion, consistent with our hypothesized model for this interspecies microbial cell fusion, we observed differential regulation of genes involved in C. ljungdahlii’s autotrophic Wood-Ljungdahl pathway metabolism and genes of the motility machinery. Unexpectedly, we also identified differential regulation of biosynthetic genes of several amino acids, and notably of arginine and histidine. We verified that they are produced by C. acetobutylicum and are metabolized by C. ljungdahlii to its growth advantage. These and other findings, and notably upregulation of C. acetobutylicum ribosomal-protein genes, paint a more complex syntrophic picture and suggest a mutualistic relationship, whereby beyond CO 2 and H 2 , C. acetobutylicum feeds C. ljungdahlii with growth-boosting amino acids, while benefiting from the H 2 utilization by C. ljungdahlii . IMPORTANCE The construction and study of synthetic microbial cocultures is a growing research area due to the untapped potential of defined multi-species industrial bioprocesses and the utility of defined cocultures for generating insight into complex, undefined, natural microbial consortia. Our previous work showed that coculturing C. acetobutylicum and C. ljungdahlii leads to a unique metabolic phenotype (production of isopropanol) and heterologous cell fusion events. Here, we used RNAseq to explore genes involved in and impacted by these fusions. First, we compared gene expression in coculture to each monoculture. Second, we utilized a transwell system to compare gene expression in mixed cocultures to cocultures with both species physically separated by a permeable membrane, isolating the impact of interspecies “touching” on the transcriptome. This study deepens our mechanistic understanding of the C. acetobutylicum-C. ljungdahlii coculture phenotype, laying the groundwork for reverse genetic studies of heterologous cell fusion in Clostridium cocultures.

Willis, Noah B. (ORCID:0009000689365955)↗
DOE OSTI2020

Dynamic resource allocation drives growth under nitrogen starvation in eukaryotes

Cells can sense changes in their extracellular environment and subsequently adapt their biomass composition. Nutrient abundance defines the capability of the cell to produce biomass components. Under nutrient-limited conditions, resource allocation dramatically shifts to carbon-rich molecules. Here, we used dynamic biomass composition data to predict changes in growth and reaction flux distributions using the available genome-scale metabolic models of five eukaryotic organisms (three heterotrophs and two phototrophs). We identified temporal profiles of metabolic fluxes that indicate long-term trends in pathway and organelle function in response to nitrogen depletion. Surprisingly, our calculations of model sensitivity and biosynthetic cost showed that free energy of biomass metabolites is the main driver of biosynthetic cost and not molecular weight, thus explaining the high costs of arginine and histidine. We demonstrated how metabolic models can accurately predict the complexity of interwoven mechanisms in response to stress over the course of growth.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Natural Product Gene Clusters in the Filamentous Nostocales Cyanobacterium HT-58-2

Cyanobacteria are known as rich repositories of natural products. One cyanobacterial-microbial consortium (isolate HT-58-2) is known to produce two fundamentally new classes of natural products: the tetrapyrrole pigments tolyporphins A–R, and the diterpenoid compounds tolypodiol, 6-deoxytolypodiol, and 11-hydroxytolypodiol. The genome (7.85 Mbp) of the Nostocales cyanobacterium HT-58-2 was annotated previously for tetrapyrrole biosynthesis genes, which led to the identification of a putative biosynthetic gene cluster (BGC) for tolyporphins. Here, bioinformatics tools have been employed to annotate the genome more broadly in an effort to identify pathways for the biosynthesis of tolypodiols as well as other natural products. A putative BGC (15 genes) for tolypodiols has been identified. Four BGCs have been identified for the biosynthesis of other natural products. Two BGCs related to nitrogen fixation may be relevant, given the association of nitrogen stress with production of tolyporphins. The results point to the rich biosynthetic capacity of the HT-58-2 cyanobacterium beyond the production of tolyporphins and tolypodiols.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Data for Rewiring Yeast Metabolism for Producing 2,3-Butanediol and Two Downstream Applications: Techno-Economic Analysis and Life Cycle Assessment of Methyl Ethyl Ketone (MEK) and Agricultural Biostimulant Production

Rising concerns for sustainability and global climate change have driven the development of sustainable production pathways for biofuels and chemicals from lignocellulosic biomass via integrated biological and chemical processes. We constructed an engineered Saccharomyces cerevisiae capable of producing 2,3-butanediol (2,3-BDO) from glucose without accumulating ethanol and glycerol, which hinder downstream processing of 2,3-BDO, through extensive metabolic reprogramming. Specifically, we introduced heterologous 2,3-BDO biosynthetic enzymes and deleted the major isozymes of ethanol and glycerol biosynthetic enzymes. In addition, we introduced an NAD+ regenerating Pyruvate-Malate (PM) cycle and enhanced the NAD+ regenerating capability of the PM cycle to resolve the redox imbalance from the deletion of ethanol and glycerol production pathways. The resulting engineered yeast produced 109.9 g/L of 2,3-BDO with a productivity of 1.0 g/L/h and a yield of 0.36 g/g glucose in a fed-batch fermentation. We also conducted techno-economic analysis (TEA) and life cycle assessment (LCA) of the production of methyl ethyl ketone (MEK) through catalytic dehydration of 2,3-BDO. A TEA based on the experimental results indicated that the minimum product selling price (MPSP) was estimated to be $1.90/kg. Regarding cradle-to-grave LCA, 100-year global warming potential (GWP100) and fossil energy consumption (FEC) were found to be 0.37 kg CO2 eq/kg and 3.1 MJ/kg, respectively. These results demonstrated the feasibility of cost-competitive and sustainable bio-based MEK production via yeast fermentation. In addition, we explored the possibility of using the fermentation broth containing 2,3-BDO as a biostimulant inducing drought tolerance in plants. As a result, the yeast 2,3-BDO fermentation broth can induce drought tolerance in Arabidopsis thaliana without a complicated purification process.

Economics↗
DOE OSTI2026

Engineered plants for the production of the antioxidants arbutin and gallate

The shikimate pathway is a crucial metabolic route for the biosynthesis of numerous valuable chemicals. In this study, we engineered the shikimate pathway in plants via expression of microbial enzymes to produce the two important antioxidants gallate and arbutin. The engineered pathways utilize the aromatics protocatechuate and 4-hydroxybenzoate as metabolic intermediates. Through transient expression in Nicotiana benthamiana leaves, we first identified biosynthetic routes for the production of gallate from either chorismate or 3-dehydroshikimate. Gallate production was then achieved in Arabidopsis using a genetic background that overproduces protocatechuate and via expression of a mutated version of the 4-hydroxybenzoate hydroxylase PobA from Pseudomonas sp. Arbutin production was obtained in Arabidopsis using a genetic background that overproduces 4-hydroxybenzoate and via expression of the monooxygenase MNX1 from Candida parapsilosis. The best Arabidopsis transgenic lines accumulated gallate and arbutin in the range of 0.25 and 0.93 dry weight % (dwt%), respectively. Using sorghum for large-scale in planta production, the titers of gallate and arbutin produced from the intermediate 4-hydroxybenzoate reached 0.58 dwt% and 0.50 dwt%, respectively, in mature transgenic plants, surpassing levels typically observed in plants that naturally produce these compounds. Gallate and arbutin were readily extracted from plant tissues using methanol solvent. Analysis of extractive-free biomass showed only trace amounts of gallate and its precursors 4-hydroxybenzoate and protocatechuate crosslinked to cell walls, suggesting that they mainly occur as soluble conjugated forms stored in the vacuole. This study presents alternative synthesis routes using plant hosts for the eco-friendly production of gallate and arbutin.

Antioxidant↗
DOE OSTI2022

Engineering of non-model eukaryotes for bioenergy and biochemical production

The prospect of leveraging naturally occurring phenotypes to overcome bottlenecks constraining the bioeconomy has marshalled increased exploration of nonconventional organisms. This review discusses the status of non-model eukaryotic species in bioproduction, the evaluation criteria for effectively matching a candidate host to a biosynthetic process, and the genetic engineering tools needed for host domestication. Here, we present breakthroughs in genome editing and heterologous pathway design, delving into innovative spatiotemporal modulation strategies that potentiate more refined engineering capabilities. We cover current understanding of genetic instability and its ramifications for industrial scale-up, highlighting key factors and possible remedies. Finally, we propose future opportunities to expand the current collection of available hosts and provide guidance to benefit the broader bioeconomy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Prolonging photoperiod promotes testosterone synthesis of Leydig cells by directly targeting local melatonin system in rooster testes

Abstract The study investigated the effects of prolonging photoperiod on the synthesis of testosterone and melatonin in roosters, and the effect of melatonin on testosterone synthesis in rooster Leydig cells as well as its molecular mechanisms. We randomly divided one hundred and twenty 20-week-old roosters into three groups and provided 6, 12.5 and 16 h light, respectively. The results showed that prolonging photoperiod promoted testosterone synthesis, decreased melatonin production, and inhibited the expression of melatonin membrane receptors MEL1A, MEL1B, MEL1C, and aralkylamine n-acetyltransferase (AANAT) in rooster testes. Subsequently, rooster Leydig cells were isolated and treated with 0, 0.1, 1, 10, and 100 ng/mL melatonin for 36 h. The results suggested that melatonin inhibited testosterone synthesis in rooster Leydig cells, and silencing MEL1A and MEL1B relieved the inhibition of melatonin on testosterone synthesis. Additionally, melatonin reduced the intracellular cyclic adenosine monophosphate (cAMP) level and the phosphorylation level of cAMP-response element binding protein (CREB), and CREB overexpression alleviated the inhibition of melatonin on testosterone synthesis. Furthermore, pretreatment with cAMP activator forskolin or protein kinase A (PKA) activator 8-bromo-cAMP blocked the inhibition of melatonin on CREB phosphorylation and testosterone synthesis. These results indicated that prolonging photoperiod promoted testosterone synthesis associated with the decrease in melatonin production and membrane receptors and biosynthetic enzyme of melatonin in rooster testes, and melatonin inhibited testosterone synthesis of rooster Leydig cells by inhibiting the cAMP/PKA/CREB pathway via MEL1A and MEL1B. This may be evidence that prolonging photoperiod could promote testosterone synthesis through the inhibition of the local melatonin pathway in rooster testes.

Xu, Gaoqing↗