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At least 307 records · Page 17
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Phenylpropanoid methyl esterase unlocks catabolism of aromatic biological nitrification inhibitors

Microbial nitrification of fertilizers represents is a significant global source of greenhouse gas emissions. This process increases emissions, fosters toxic algal blooms, and raises crop production costs. Some plants naturally release biological nitrification inhibitors to suppress ammonium-oxidizing microbes and reduce nitrification. Engineering nitrification inhibitor production into food and bioenergy crops via synthetic biology offers a promising mitigation strategy, but its success depends on addressing gaps in our understanding of inhibitor degradation in soil. This study begins to fill this gap by identifying a previously unknown microbial pathway for degrading phenylpropanoid methyl esters, a key class of aromatic nitrification inhibitors. Using transcriptomics and high-throughput functional genomics, we discovered genes essential for phenylpropanoid methyl ester degradation. Genetic and biochemical analyses revealed two novel enzymes, including a newly identified phenylpropanoid methyl esterase, that direct phenylpropanoid methyl esters into known metabolic pathways. Importantly, transferring these genes into bacteria capable of metabolizing other phenylpropanoids enabled them to use the methyl esters as a carbon source. This work provides critical insights into microbial nitrification inhibitor degradation, a poorly understood element of the nitrification cycle.

Genetic Engineering↗
DOE OSTI2022

Plant-based engineering for production of high-valued natural products

Plants are a unique source of complex specialized metabolites, many of which play significant roles in human society. In many cases, however, the availability of these metabolites from naturally occurring sources fails to meet current demands. Thus, there is much interest in expanding the production capacity of target plant molecules. Traditionally, plant breeding, chemical synthesis, and microbial fermentation are considered the primary routes towards large scale production of natural products. Here, in this paper, we explore the advances, challenges, and future of plant engineering as a complementary path. Although plants are an integral part of our food and agricultural systems and sustain an extensive array of chemical constituents, their complex genetics and physiology have prevented the optimal exploitation of plants as a production chassis. We highlight emerging engineering tools and scientific advances developed in recent years that have improved the prospects of using plants as a sustainable and scalable production platform. We also discuss technological limitations and overall economic outlook of plant-based production of natural products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Advancing biological processing for valorization of plastic wastes

We report the useful applications of plastics are as significant as the problems associated with their disposal. Current waste management strategies are ineffective at preventing the flow of plastics into landfills, wastewater treatment, and the environment. Despite the importance of recycling technologies, most plastics are used for consumables and are discarded after limited usage. Moreover, only certain plastics are recycled, and those that are generally yield lower value products. Discovering alternative routes for valorizing plastic wastes is essential. Emerging research involves the use of biocatalysts (i.e., enzymes and microorganisms) to produce valuable products like specialty polymers, biosurfactants, and drug precursors from these feedstocks. These bioprocesses commonly integrate physio-chemical pretreatments (to generate biologically reactive intermediates) and biological upgrading (to convert intermediates to products). Herein, publications that explore plastic deconstruction technologies and bioprocesses are reviewed with an emphasis on plastic waste valorization. Understanding how microorganisms metabolize the intermediates of plastic degradation and manage their inhibitory effects is a prerequisite for engineering bioprocesses. For these heterogeneous wastes, a paradigm shift from axenic cultivation of microorganisms to cultivation of microbial consortia may benefit conversion efficiency and stress resilience. Thus, a summary is provided for metabolic pathways and constituent enzymes required to synthesize biofuels, biomaterials, and chemicals from biologically reactive intermediates. Colossal challenges associated with the use of plastics as feedstocks for direct biological processing highlight the need for integrating physical, chemical, and biological technologies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Investigating the eco‐evolutionary response of microbiomes to environmental change

Abstract Microorganisms are the primary engines of biogeochemical processes and foundational to the provisioning of ecosystem services to human society. Free‐living microbial communities (microbiomes) and their functioning are now known to be highly sensitive to environmental change. Given microorganisms' capacity for rapid evolution, evolutionary processes could play a role in this response. Currently, however, few models of biogeochemical processes explicitly consider how microbial evolution will affect biogeochemical responses to environmental change. Here, we propose a conceptual framework for explicitly integrating evolution into microbiome–functioning relationships. We consider how microbiomes respond simultaneously to environmental change via four interrelated processes that affect overall microbiome functioning (physiological acclimation, demography, dispersal and evolution). Recent evidence in both the laboratory and the field suggests that ecological and evolutionary dynamics occur simultaneously within microbiomes; however, the implications for biogeochemistry under environmental change will depend on the timescales over which these processes contribute to a microbiome's response. Over the long term, evolution may play an increasingly important role for microbially driven biogeochemical responses to environmental change, particularly to conditions without recent historical precedent.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format

Plate-based proteomic sample preparation offers a solution to the large sample throughput demands in the biotechnology field where hundreds or thousands of engineered microbes are constructed for testing is routine. Meanwhile, sample preparation methods that work efficiently on broader microbial groups are desirable for new applications of proteomics in other fields, such as microbial communities. Here, we detail a step-by-step protocol that consists of cell lysis in an alkaline chemical buffer (NaOH/SDS) followed by protein precipitation with high-ionic strength acetone in 96-well format. The protocol works for a broad range of microbes ( e . g ., Gram-negative bacteria, Gram-positive bacteria, non-filamentous fungi) and the resulting proteins are ready for tryptic digestion for bottom-up quantitative proteomic analysis without the need for desalting column cleanup. The yield of protein using this protocol increases linearly with respect to the amount of starting biomass from 0.5–2.0 OD*mL of cells. By using a bench-top automated liquid dispenser, a cost-effective and environmentally-friendly option to eliminating pipette tips and reducing reagent waste, the protocol takes approximately 30 minutes to extract protein from 96 samples. Tests on mock mixtures showed expected results that the biomass composition structure is in close agreement with the experimental design. Lastly, we applied the protocol for the composition analysis of a synthetic community of environmental isolates grown on two different media. This protocol has been developed to facilitate rapid, low-variance sample preparation of hundreds of samples and allow flexibility for future protocol development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Heterologous expression of formate dehydrogenase enables photoformatotrophy in the emerging model microalga, Picochlorum renovo

Rising global greenhouse gas emissions and the impacts of resultant climate change necessitate development and deployment of carbon capture and conversion technologies. Amongst the myriad of bio-based conversion approaches under evaluation, a formate bio-economy has recently been proposed, wherein CO 2 -derived formate serves as a substrate for concurrent carbon and energy delivery to microbial systems. To date, this approach has been explored in chemolithotrophic and heterotrophic organisms via native or engineered formatotrophy. However, utilization of this concept in phototrophic organisms has yet to be reported. Herein, we have taken the first steps to establish formate utilization in Picochlorum renovo, a recently characterized eukaryotic microalga with facile genetic tools and promising applied biotechnology traits. Plastidial heterologous expression of a formate dehydrogenase (FDH) enabled P. renovo growth on formate as a carbon and energy source. Further, FDH expression enhanced cultivation capacity on ambient CO 2 , underscoring the potential for bypass of conventional CO 2 capture and concentration limitations. This work establishes a photoformatotrophic cultivation regime that leverages light energy-driven formate utilization. The resultant photosynthetic formate platform has widespread implications for applied phototrophic cultivation systems and the bio-economy at large.

09 BIOMASS FUELS↗
NASA NTRS2000

Morphology and Viability of Pleistocene Microbiota from the CRREL Permafrost Tunnel Near Fox, Alaska

The U. S. Army Cold Regions Research and Engineering Laboratory maintains the CRREL Permafrost Tunnel at Fox, Alaska (-10 miles north of Fairbanks.) The active microbial ecosystems and the cryopreserved anabiotic viable microorganisms and dead microbial remains and biomarkers frozen within the permafrost and ice of the CRREL Permafrost Tunnel are of direct relevance to Astrobiology. Microbial extremophiles from permafrost and ice provide information concerning where and how should we search for evidence of life elsewhere in the Cosmos. The permafrost and ice wedges of the Fox tunnel preserves a magnificent of record of Pliocene, Pleistocene and Holocene life on Earth spanning more than 2.5 million years. This record includes frozen fossil bacteria, archaea, algae, mosses, higher plants, insects and mammals. In this paper we present the preliminary results of studies of the morphology, ultramicrostructure and elemental distributions of Fox tunnel microbiota as determined in-situ by the Environmental Scanning Electron Microscope (ESEM) and the Field Emission Scanning Electron Microscope (FESEM) investigations. The long-term viability of cryopreserved microbiota and potential implications to Astrobiology will be discussed.

Hoover, Richard B.↗
DOE OSTI2024

Assessing horizontal gene transfer in the rhizosphere of Brachypodium distachyon using fabricated ecosystems (EcoFABs)

ABSTRACT Horizontal gene transfer (HGT) is a major process by which genes are transferred between microbes in the rhizosphere. However, examining HGT remains challenging due to the complexity of mimicking conditions within the rhizosphere. Fabricated ecosystems (EcoFABs) have been used to investigate several complex processes in plant-associated environments. Here we show that EcoFABs are efficient tools to examine and measure HGT frequency in the rhizosphere. We provide the first demonstration of gene transfer via a triparental conjugation system in the Brachypodium distachyon rhizosphere in an EcoFAB using Pseudomonas putida KT2440 as both donor and recipient bacterial strain with the donor containing a mobilizable and non-self-transmissible plasmid. We observed that the frequency of plasmid transfer in the rhizosphere is potentially dependent on the plant developmental stage and the composition and amount of root exudates. The frequency of plasmid transfer also increased with higher numbers of donor cells. We demonstrate the transfer of plasmid from P. putida to another B. distachyon root colonizer, Burkholderia sp. OAS925, showing HGT within a rhizosphere microbial community. Environmental stresses also influenced the rate and efficiency of HGT in the rhizosphere between different species and genera. This study provides a robust workflow to evaluate transfer of engineered plasmids in the rhizosphere when such plasmids are potentially introduced in a field or other plant-associated environments. IMPORTANCE We report the use of EcoFABs to investigate the HGT process in a rhizosphere environment. It highlights the potential of EcoFABs in recapitulating the dynamic rhizosphere conditions as well as their versatility in studying plant-microbe interactions. This study also emphasizes the importance of studying the parameters impacting the HGT frequency. Several factors such as plant developmental stages, nutrient conditions, number of donor cells, and environmental stresses influence gene transfer within the rhizosphere microbial community. This study paves the way for future investigations into understanding the fate and movement of engineered plasmids in a field environment.

Priya, Shweta↗
DOE OSTI2026

Finding the missing pieces: filling gaps that impede the translation of omics data into models

High-throughput omics technologies such as DNA sequencing have made the sequencing and computational assembly of microbial genomes recovered from the environment relatively routine. Computational inference of the protein products encoded by these genomes, and the associated biochemical functions, should enable the accurate prediction and modeling of microbial metabolism, organismal interactions, and ecosystem processes. However, a lack of scalable, probabilistic protein annotation tools limits the full potential of modeling for understanding the metabolism and biogeochemical cycles of microbial communities. Our approach to improve inference of protein annotations and metabolic models relied on learning from and emulating expert manual curation, leveraging software engineering and data science best practices to scale up the throughput and accuracy of annotations and metabolic model construction, building software to objectively evaluate different annotation strategies, and more closely linking the protein annotation and metabolic model inference process. Outcomes of this research include several improved or new computational tools, including DRAM (Distilled and Refined Annotation of Metabolism) for annotating microbial genomes with protein function and metabolic traits, CAMPER (Curated Annotations for Microbial Polyphenol Enzymes and Reactions) for annotating key polyphenol metabolisms, EC-Bench for comprehensive and unbiased benchmarking of annotation tools, and several apps available via the DOE Systems Biology Knowledgebase (KBase) for building genome-scale metabolic models. We demonstrate that these tools allow us to scalably annotate and understand thousands of genomes for microbial communities from a variety of systems and test cases, including rivers, thawing permafrost, and gut microbiomes. All of these computational tools are available as open-source software, with most broadly and easily accessible to the scientific community via KBase apps.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

2024 International Conference on Microbiome Engineering (ICME)

The 2024 International Conference on Microbiome Engineering (ICME) took place November 12-14 at Tufts University in Medford, MA. ICME connects experts from academia and industry to share the most recent developments in the field of microbiome engineering. This includes genetically engineered organisms that function within microbiomes, control of microbiomes through environmental/nutrient modifications, and inference of engineering principles from analysis of synthetic and natural microbiomes. The conference is unique and distinct from other microbiome conferences in that it specifically highlights the integration of engineering design principles with microbiome research (others are more focused on basic biological principles). The conference thus integrates synthetic biology, systems biology, microbial ecology, and bioinformatics across a range of application spaces from the environment to manufacturing, food, and human health. This project utilized support from the Department of Energy’s (DOE) Office of Biological and Environmental Research (BER) to help trainees and early career faculty attend ICME.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Improving growth of Cupriavidus necator H16 on formate using adaptive laboratory evolution-informed engineering

Conversion of CO 2 to value-added products presents an opportunity to reduce GHG emissions while generating revenue. Formate, which can be generated by the electrochemical reduction of CO 2 , has been proposed as a promising intermediate compound for microbial upgrading. Here we present progress towards improving the soil bacterium Cupriavidus necator H16, which is capable of growing on formate as its sole source of carbon and energy using the Calvin-Benson-Bassham (CBB) cycle, as a host for formate utilization. Using adaptive laboratory evolution, we generated several isolates that exhibited faster growth rates on formate. The genomes of these isolates were sequenced, and resulting mutations were systematically reintroduced by metabolic engineering, to identify those that improved growth. Further, the metabolic impact of several mutations was investigated further using RNA-seq transcriptomics. We found that deletion of a transcriptional regulator implicated in quorum sensing, PhcA, reduced expression of several operons and led to improved growth on formate. Growth was also improved by deleting large genomic regions present on the extrachromosomal megaplasmid pHG1, particularly two hydrogenase operons and the megaplasmid CBB operon, one of two copies present in the genome. Based on these findings, we generated a rationally engineered ..delta..phcA and megaplasmid-deficient strain that exhibited a 24% faster maximum growth rate on formate. Moreover, this strain achieved a 7% growth rate improvement on succinate and a 19% increase on fructose, demonstrating the broad utility of microbial genome reduction. This strain has the potential to serve as an improved microbial chassis for biological conversion of formate to value-added products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Data for Metabolic Engineering of Rhodotorula toruloides IFO0880 Improves C16 and C18 Fatty Alcohol Production from Synthetic Media

The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product. Fatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets (ACL1 and ACC1, improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides , while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption. The fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1, or the deletion of DGA1 combined with overexpression of ACC1 and ACL1. Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.

Conversion↗
NASA NTRS2011

Post-Fragmentation Whole Genome Amplification-Based Method

This innovation is derived from a proprietary amplification scheme that is based upon random fragmentation of the genome into a series of short, overlapping templates. The resulting shorter DNA strands (<400 bp) constitute a library of DNA fragments with defined 3 and 5 termini. Specific primers to these termini are then used to isothermally amplify this library into potentially unlimited quantities that can be used immediately for multiple downstream applications including gel eletrophoresis, quantitative polymerase chain reaction (QPCR), comparative genomic hybridization microarray, SNP analysis, and sequencing. The standard reaction can be performed with minimal hands-on time, and can produce amplified DNA in as little as three hours. Post-fragmentation whole genome amplification-based technology provides a robust and accurate method of amplifying femtogram levels of starting material into microgram yields with no detectable allele bias. The amplified DNA also facilitates the preservation of samples (spacecraft samples) by amplifying scarce amounts of template DNA into microgram concentrations in just a few hours. Based on further optimization of this technology, this could be a feasible technology to use in sample preservation for potential future sample return missions. The research and technology development described here can be pivotal in dealing with backward/forward biological contamination from planetary missions. Such efforts rely heavily on an increasing understanding of the burden and diversity of microorganisms present on spacecraft surfaces throughout assembly and testing. The development and implementation of these technologies could significantly improve the comprehensiveness and resolving power of spacecraft-associated microbial population censuses, and are important to the continued evolution and advancement of planetary protection capabilities. Current molecular procedures for assaying spacecraft-associated microbial burden and diversity have inherent sample loss issues at practically every step, particularly nucleic acid extraction. In engineering a molecular means of amplifying nucleic acids directly from single cells in their native state within the sample matrix, this innovation has circumvented entirely the need for DNA extraction regimes in the sample processing scheme.

Benardini, James↗
DOE OSTI2021

Diversifying Isoprenoid Platforms via Atypical Carbon Substrates and Non-model Microorganisms

Isoprenoid compounds are biologically ubiquitous, and their characteristic modularity has afforded products ranging from pharmaceuticals to biofuels. Isoprenoid production has been largely successful in Escherichia coli and Saccharomyces cerevisiae with metabolic engineering of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways coupled with the expression of heterologous terpene synthases. Yet conventional microbial chassis pose several major obstacles to successful commercialization including the affordability of sugar substrates at scale, precursor flux limitations, and intermediate feedback-inhibition. Now, recent studies have challenged typical isoprenoid paradigms by expanding the boundaries of terpene biosynthesis and using non-model organisms including those capable of metabolizing atypical C1 substrates. Conversely, investigations of non-model organisms have historically informed optimization in conventional microbes by tuning heterologous gene expression. Here, we review advances in isoprenoid biosynthesis with specific focus on the synergy between model and non-model organisms that may elevate the commercial viability of isoprenoid platforms by addressing the dichotomy between high titer production and inexpensive substrates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Metabolic engineering of Rhodotorula toruloides IFO0880 improves C16 and C18 fatty alcohol production from synthetic media

Abstract Background The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product. Results Fatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets ( ACL1 and ACC1 , improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1 ) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1 Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides , while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption. Conclusions The fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1 , or the deletion of DGA1 combined with overexpression of ACC1 and ACL1 . Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2017

Engineering of Methane Metabolism in Pichia Pastoris Through Methane Monooxygenase Expression

Exploration of the solar system is constrained by the cost of moving mass off Earth. Producing materials in situ will reduce the mass that must be delivered from earth. CO2 is abundant on Mars and manned spacecraft. On the ISS, NASA reacts excess CO2 with H2 to generate CH4 and H2O using the Sabatier System. The resulting water is recovered into the ISS, but the methane is vented to space. Thus, there is a capability need for systems that convert methane into valuable materials. Methanotrophic bacteria consume methane but these are poor synthetic biology platforms. Thus, there is a knowledge gap in utilizing methane in a robust and flexible synthetic biology platform. The yeast Pichia pastoris is a refined microbial factory that is used widely by industry because it efficiently secretes products. Pichia could produce a variety of useful products in space. Pichia does not consume methane but robustly consumes methanol, which is one enzymatic step removed from methane. Our goal is to engineer Pichia to consume methane thereby creating a powerful methane-consuming microbial factory.

Pichia↗