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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 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Wolffia, a minimalist plant and synthetic biology chassis

A highly simplified species for genome engineering would facilitate rational design of a synthetic plant. A candidate species is the aquatic, non-grass monocot wolffia (Wolffia australiana) in the Lemnaceae family. Commonly known as watermeal, wolffia is a rootless ball of several thousand cells the size of a pinhead and the fastest growing plant known on Earth. Its extreme morphological reduction is coupled to transposon-mediated streamlining of its transcriptome, which represents a core set of nonredundant protein coding genes. Despite its body plan and transcriptome being highly specialized for continuous growth, wolffia retains cell types relevant to higher plants. Finally, systems level studies with this species could enable the creation of a defined biological chassis for synthetic plant construction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A synthetic promoter system for well-controlled protein expression with different carbon sources in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an important synthetic biology chassis for microbial production of valuable molecules. Promoter engineering has been frequently applied to generate more synthetic promoters with a variety of defined characteristics in order to achieve a well-regulated genetic network for high production efficiency. Galactose-inducible (GAL) expression systems, composed of GAL promoters and multiple GAL regulators, have been widely used for protein overexpression and pathway construction in S. cerevisiae. However, the function of each element in synthetic promoters and how they interact with GAL regulators are not well known. Here, a library of synthetic GAL promoters demonstrate that upstream activating sequences (UASs) and core promoters have a synergistic relationship that determines the performance of each promoter under different carbon sources. We found that the strengths of synthetic GAL promoters could be fine-tuned by manipulating the sequence, number, and substitution of UASs. Core promoter replacement generated synthetic promoters with a twofold strength improvement compared with the GAL1 promoter under multiple different carbon sources in a strain with GAL1 and GAL80 engineering. These results represent an expansion of the classic GAL expression system with an increased dynamic range and a good tolerance of different carbon sources. In this study, the effect of each element on synthetic GAL promoters has been evaluated and a series of well-controlled synthetic promoters are constructed. By studying the interaction of synthetic promoters and GAL regulators, synthetic promoters with an increased dynamic range under different carbon sources are created.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRSDate not supplied

Developing a Genetic Variant Calling Pipeline for Quantifying the Complex Mutagenic Load Accumulated in BioNutrients-1 Production Pack Samples

Microorganisms hold great promise for on demand production of labile nutrients and pharmaceuticals as well recycling and in situ resource utilization. The utilization of microorganisms for such tasks on space missions is hindered by the limited data on how microbes respond to spaceflight. For example, the genetic stability of microorganisms, and the genomic engineered traits added to deliver desired functions, over long-term storage in the spacecraft environment is poorly understood. The BioNutrients-1 (BN-1) mission conducted a 5-year study of desiccated storage in Low Earth Orbit (LEO) to evaluate the suitability of eight synthetic biology chassis organisms for long-duration space missions. We are employing high-depth, whole genome sequencing (WGS) to determine the mutagenic load that accumulated during long-term storage. Mutation analysis pipelines are well established for homogenous culture grown from a single colony, but the mutational landscape of the BN-1 samples present a unique analysis challenge, as every cell in the BN-1 samples had a unique genetic journey of DNA damage and repair. Consequently, sequence variants are expected at low allele frequency within samples. To address this genetic complexity, we apply two distinct computational approaches to identify mutations in pre-existing WGS data collected from populations of Chlamydomonas reinhardtii that were exposed to UV mutagenesis and growth in LEO. For reference genome free mutation detection, we utilized DiscoSNP++, which is a de Bruijn graph approach. For reference genome-based mutation detection we utilize GATK for Microbes, which is a Bayesian probabilistic approach. We will benchmark these approaches against the mutations originally identified using CRISP, a method optimized for pooled samples. Ultimately, quantifying the mutation load imposed by storage or growth on the ISS will help identify chassis organisms with both high levels of genome stability and viability, which are desirable traits for implementation of bioproduction in long-duration missions.

SNP↗
DOE OSTI2023

Genome sequence and characterization of a novel Pseudomonas putida phage, MiCath

Abstract Pseudomonads are ubiquitous bacteria with importance in medicine, soil, agriculture, and biomanufacturing. We report a novel Pseudomonas putida phage, MiCath, which is the first known phage infecting P. putida S12, a strain increasingly used as a synthetic biology chassis. MiCath was isolated from garden soil under a tomato plant using P. putida S12 as a host and was also found to infect four other P. putida strains. MiCath has a ~ 61 kbp double-stranded DNA genome which encodes 97 predicted open reading frames (ORFs); functions could only be predicted for 48 ORFs using comparative genomics. Functions include structural phage proteins, other common phage proteins (e.g., terminase), a queuosine gene cassette, a cas4 exonuclease, and an endosialidase. Restriction digestion analysis suggests the queuosine gene cassette encodes a pathway capable of modification of guanine residues. When compared to other phage genomes, MiCath shares at most 74% nucleotide identity over 2% of the genome with any sequenced phage. Overall, MiCath is a novel phage with no close relatives, encoding many unique gene products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Beyond Component Optimization: Systems Level Biodesign for Lanthanide Recovery

Global demand for lanthanides (Ln) is projected to rise sharply over the next decade, while geographically concentrated supply chains and the low concentrations and matrix complexity of secondary feedstocks limit the reach of conventional hydro- and pyrometallurgical separation. Engineered biological systems offer a selective, low-energy alternative, and component-level advances in Ln-binding proteins, AI-designed selective scaffolds, and cell-surface display platforms now rival synthetic chelators in affinity and selectivity. These components, however, remain functionally isolated. Currently, there are no engineered chassis coupling recognition, intracellular trafficking, accumulation, and controlled release into an end-to-end pipeline. Here, we outline how new biodesign strategies and chassis selection must move beyond bioleaching to encompass the full recovery pathway. Achieving this requires integrating AI/ML-guided design, genome-scale build tools, high-throughput phenotyping, and biophysical transport modeling within a Design–Build–Test–Learn cycle tuned to recognition, trafficking, accumulation, and release.

Biodesign↗
DOE OSTI2024

Optimized CRISPR Interference System for Investigating Pseudomonas alloputida Genes Involved in Rhizosphere Microbiome Assembly

Pseudomonas alloputida KT2440 (formerly P. putida) has become both a well-known chassis organism for synthetic biology and a model organism for rhizosphere colonization. Here, we describe a CRISPR interference (CRISPRi) system in KT2440 for exploring microbe–microbe interactions in the rhizosphere and for use in industrial systems. Our CRISPRi system features three different promoter systems (XylS/P m , LacI/P lac , and AraC/P BAD ) and a dCas9 codon-optimized for Pseudomonads, all located on a mini-Tn7-based transposon that inserts into a neutral site in the genome. It also includes a suite of pSEVA-derived sgRNA expression vectors, where the expression is driven by synthetic promoters varying in strength. We compare the three promoter systems in terms of how well they can precisely modulate gene expression, and we discuss the impact of environmental factors, such as media choice, on the success of CRISPRi. We demonstrate that CRISPRi is functional in bacteria colonizing the rhizosphere, with repression of essential genes leading to a 10–100-fold reduction in P. alloputida cells per root. Finally, we show that CRISPRi can be used to modulate microbe–microbe interactions. When the gene pvdH is repressed and P. alloputida is unable to produce pyoverdine, it loses its ability to inhibit other microbes in vitro. Furthermore, our design is amendable for future CRISPRi-seq studies and in multispecies microbial communities, with the different promoter systems providing a means to control the level of gene expression in many different environments.

Bacteria↗
NASA NTRS2019

Results of the Micro-12 Flight Experiment: Effects of Microgravity on Shewanella oneidensis MR-1

The Micro-12 flight experiment was launched on SpaceX-15 and completed during berthing on the International Space Station. The goal of this experiment was to understand the effects of spaceflight and microgravity on the physiology of the model exoelectrogen Shewanella oneidensis MR-1. BioServe Fluid Processing Apparatus (FPA) and Group Activation Pack (GAP) hardware systems were used for both flight and ground control tests. Under spaceflight conditions, extracellular electron transfer (EET) rates were found to be significantly increased on insoluble substrates, while biofilm development appeared to be unchanged under the conditions tested; these processes are critical for microbial-assisted bioelectrochemical systems. Additionally, RNAseq analysis, proteomic profiling, and competitive mutant fitness profiling were performed to gain further understanding of microbial physiology under EET-respiring conditions during spaceflight. Overall, the results of the Micro-12 project support the idea that Shewanella oneidensis MR-1, in particular, and exoelectrogens in general could be useful chassis organisms for synthetic biology applications using microbial bioelectrochemical systems. These findings will assist bioengineering and synthetic biology development efforts harnessing the unique capabilities of exoelectrogens for life support and in situ resource utilization.

Dougherty, Michael↗
DOE OSTI2024

Producing multiple chemicals through biological upcycling of waste poly(ethylene terephthalate)

Poly(ethylene terephthalate) (PET) waste is of low degradability in nature, and its mismanagement threatens numerous ecosystems. To combat the accumulation of waste PET in the biosphere, PET bio-upcycling, which integrates chemical pretreatment to produce PET-derived monomers with their microbial conversion into value-added products, has shown promise. The recently discovered Rhodococcus jostii strain PET (RPET) can metabolically degrade terephthalic acid (TPA) and ethylene glycol (EG) as sole carbon sources, and it has been developed into a microbial chassis for PET upcycling. However, the scarcity of synthetic biology tools, specifically designed for the non-model microbe RPET, limits the development of a microbial cell factory for expanding the repertoire of bioproducts from post-consumer PET. Herein, we describe the development of potent genetic tools for RPET, including (1) two inducible and titratable expression systems for tunable gene expression and (2) Serine Integrase-based Recombinational Tools (SIRT) for genome editing. Using these tools, we systematically engineer the RPET strain to ultimately establish microbial supply chains for producing multiple chemicals, including lycopene, lipids, and succinate, from post-consumer PET waste bottles, achieving the highest titer of lycopene ever reported thus far in RPET (i.e., 22.6 mg/L of lycopene, approximately 10,000-fold higher than that of the wild-type strain). Furthermore, this work highlights the great potential of plastic upcycling as a generalizable means of sustainable production of diverse chemicals.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Bacterial microcompartments as a next-generation metabolic engineering tool: utilizing nature's solution for confining challenging catabolic pathways

Advancements in synthetic biology have facilitated the incorporation of heterologous metabolic pathways into various bacterial chassis, leading to the synthesis of targeted bioproducts. However, total output from heterologous production pathways can suffer from low flux, enzyme promiscuity, formation of toxic intermediates, or intermediate loss to competing reactions, which ultimately hinder their full potential. The self-assembling, easy-to-modify, protein-based bacterial microcompartments (BMCs) offer a sophisticated way to overcome these obstacles by acting as an autonomous catalytic module decoupled from the cell's regulatory and metabolic networks. More than a decade of fundamental research on various types of BMCs, particularly structural studies of shells and their self-assembly, the recruitment of enzymes to BMC shell scaffolds, and the involvement of ancillary proteins such as transporters, regulators, and activating enzymes in the integration of BMCs into the cell's metabolism, has significantly moved the field forward. These advances have enabled bioengineers to design synthetic multi-enzyme BMCs to promote ethanol or hydrogen production, increase cellular polyphosphate levels, and convert glycerol to propanediol or formate to pyruvate. These pioneering efforts demonstrate the enormous potential of synthetic BMCs to encapsulate non-native multi-enzyme biochemical pathways for the synthesis of high-value products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Unlocking the magic in mycelium: Using synthetic biology to optimize filamentous fungi for biomanufacturing and sustainability

Filamentous fungi drive carbon and nutrient cycling across our global ecosystems, through its interactions with growing and decaying flora and their constituent microbiomes. The remarkable metabolic diversity, secretion ability, and fiber-like mycelial structure that have evolved in filamentous fungi have been increasingly exploited in commercial operations. The industrial potential of mycelial fermentation ranges from the discovery and bioproduction of enzymes and bioactive compounds, the decarbonization of food and material production, to environmental remediation and enhanced agricultural production. Despite its fundamental impact in ecology and biotechnology, molds and mushrooms have not, to-date, significantly intersected with synthetic biology in ways comparable to other industrial cell factories (e.g. Escherichia coli,Saccharomyces cerevisiae, and Komagataella phaffii). In this review, we summarize a suite of synthetic biology and computational tools for the mining, engineering and optimization of filamentous fungi as a bioproduction chassis. A combination of methods across genetic engineering, mutagenesis, experimental evolution, and computational modeling can be used to address strain development bottlenecks in established and emerging industries. These include slow mycelium growth rate, low production yields, non-optimal growth in alternative feedstocks, and difficulties in downstream purification. In the scope of biomanufacturing, we then detail previous efforts in improving key bottlenecks by targeting protein processing and secretion pathways, hyphae morphogenesis, and transcriptional control. Bringing synthetic biology practices into the hidden world of molds and mushrooms will serve to expand the limited panel of host organisms that allow for commercially-feasible and environmentally-sustainable bioproduction of enzymes, chemicals, therapeutics, foods, and materials of the future.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

An argument for using anaerobes as microbial cell factories to advance synthetic biology and biomanufacturing

Anaerobes thrive in the absence of oxygen and are an untapped reservoir of biotechnological potential. Therefore, bioprospecting efforts focused on anaerobic microbial diversity could rapidly uncover new enzymes, pathways, and chassis organisms to drive biotechnology innovation. Despite their potential utility, anaerobic fermenters are viewed as inefficient from a biochemical perspective because their metabolisms produce fewer ATP (~2) per molecule of glucose processed than heterotrophic respirers (~32–38 ATP). While aerobes excel at ATP generation, they are often less efficient than anaerobes at processes that compete with ATP generation for cellular resources. This perspective highlights how anaerobic adaptations are advantageous for synthetic biology and biomanufacturing applications through the engineering of microbial cell factories. We further highlight emerging applications of anaerobic bioprocessing, including the use of anaerobic metabolisms for lignocellulosic bioprocessing, human and environmental health, and value-added bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Building an expanded bio-based economy through synthetic biology

The field of synthetic biology is essential to the continued development of a bio-based economy, creating mechanisms to supply carbon needed in the economy by both converting existing end-of-life wastes as well as by creating novel, purpose-grown and sustainable feedstocks. Here, we first discuss the near- and long-term resources available for use as feedstocks for bioconversion as well as the output molecules needed for building the foundation of an expanded bio-based economy. We then outline the organisms and phenotypic traits that are needed for the performance-advantaged chassis organisms of the future. Furthermore, we detail the advances, challenges, and opportunities in both microbial and plant synthetic biology relevant to expanding the bio-based economy. Finally, we explore technologies that have and will further enable advances in synthetic biology and the greater bio-based economy.

09 BIOMASS FUELS↗
DOE OSTI2025

Through the lens of bioenergy crops: advances, bottlenecks, and promises of plant engineering

Advances in engineering of bioenergy crops were driven over the past years by adapting technological breakthroughs and accelerating conventional applications but also exposed intriguing challenges. New tools revealed rich interconnectivity in the exponentially growing and dynamic 'big' omics data' of metabolomes, transcriptomes, and genomes at previously inaccessible magnitude (global, cross-species, meta-) and resolution (single cell). Insights enabled fresh hypotheses and stimulated disciplines such as functional genomics with discovery of broad regulatory networks and their determinants, that is, DNA parts, including promoters, regulatory elements, and transcription factors. Their rational design, assembly into increasingly complex blueprints, and installation into diverse chassis is an existing frontier that may benefit from emerging technologies to address bottlenecks. Interweaving nature-inspired to fully synthetic parts has already allowed building of fine-tuned regulatory circuits, or new-to-nature metabolic routes insulated from the biological context of the chassis species. Similarly, developments and the evolving need for unifying principles in plant transformation and species-agnostic technologies highlight future opportunities for engineering the next generation of bioenergy plants.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2013

Cosmo Cassette: A Microfluidic Microgravity Microbial System For Synthetic Biology Unit Tests and Satellite Missions

Although methods in the design-build-test life cycle of the synthetic biology field have grown rapidly, the expansion has been non-uniform. The design and build stages in development have seen innovations in the form of biological CAD and more efficient means for building DNA, RNA, and other biological constructs. The testing phase of the cycle remains in need of innovation. Presented will be both a theoretical abstraction of biological measurement and a practical demonstration of a microfluidics-based platform for characterizing synthetic biological phenomena. Such a platform demonstrates a design of additive manufacturing (3D printing) for construction of a microbial fuel cell (MFC) to be used in experiments carried out in space. First, the biocompatibility of the polypropylene chassis will be demonstrated. The novel MFCs will be cheaper, and faster to make and iterate through designs. The novel design will contain a manifold switchingdistribution system and an integrated in-chip set of reagent reservoirs fabricated via 3D printing. The automated nature of the 3D printing yields itself to higher resolution switching valves and leads to smaller sized payloads, lower cost, reduced power and a standardized platform for synthetic biology unit tests on Earth and in space. It will be demonstrated that the application of unit testing in synthetic biology will lead to the automatic construction and validation of desired constructs. Unit testing methodologies offer benefits of preemptive problem identification, change of facility, simplicity of integration, ease of documentation, and separation of interface from implementation, and automated design.

Bioelectrochemical System↗
DOE OSTI2025

A framework for challenges and solutions in biodesign research

The bioeconomy represents an advanced economic paradigm that builds upon previous agricultural, industrial, and digital economic models. It seeks to tackle critical global challenges such as resource scarcity, escalating healthcare demands, and environmental degradation. At the heart of the bioeconomy is biomanufacturing, which uses natural or engineered enzymes or cell factories built from ​biological components like promoters, terminators, regulatory sequences, reporters, and functional genes into various chassis hosts (including animal, microbial, plant, and de novo systems) to create products such as food, energy, medicine, materials, chemicals, and engineered tissue/organs. An enabler of biomanufacturing is biodesign – also known as biosystems design and closely related to synthetic biology or engineering biology. This interdisciplinary field aims to understand and predictably modify existing life forms or create entirely new biological entities/systems using rational engineering strategies and automated design tools. Through these capabilities, biodesign supports the discovery, optimization, and creation of efficient platforms for biomanufacturing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A FAIR-compliant parts catalogue for genome engineering and expression control in Saccharomyces cerevisiae

The synthetic biology toolkit for baker's yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A broad-host-range event detector: expanding and quantifying performance between Escherichia coli and Pseudomonas species

Modern microbial biodesign relies on the principle that well-characterized genetic parts can be reused and reconfigured for different functions. However, this paradigm has only been successful in a limited set of hosts, mostly comprised from common lab strains of Escherichia coli . It is clear that new applications such as chemical sensing and event logging in complex environments will benefit from new host chassis. This study quantitatively compared how the same chemical event logger performed across four strains and three different microbial species. An integrase-based sensor and memory device was operated by two representative soil Pseudomonads— Pseudomonas fluorescens SBW25 and Pseudomonas putida DSM 291. Quantitative comparisons were made between these two non-traditional hosts and two benchmark E. coli chassis including the probiotic Nissle 1917 and common cloning strain DH5α. The performance of sensor and memory components changed according to each host, such that a clear chassis effect was observed and quantified. These results were obtained via fluorescence from reporter proteins that were transcriptionally fused to the integrase and downstream recombinant region and via data-driven kinetic models. The Pseudomonads proved to be acceptable chassis for the operation of this event logger, which outperformed the common E. coli DH5α in many ways. This study advances an emerging frontier in synthetic biology that aims to build broad-host-range devices and understand the context by which different species can execute programmable genetic operations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Microbial spies and bloggers: programming cells to convert environmental information into discernible signals

Microbes regulate their dynamic behaviors using the chemical and physical characteristics of their environment. The ability of microbes to continuously convert this physicochemical information into biochemical information and to use organic matter in the environment as a power source makes these organisms attractive as chassis for building sensors. However, most biosensors have severe limitations when considering applications in hard-to-image settings like soils, sediments, and wastewater. Emerging technologies at the interface of biomolecular design, microbiome engineering, and synthetic biology offer new tools to program cells and communities as biosensors for these settings. Here, in this review, we describe innovations in biosensor outputs that are enabling new applications in complex environments, including reporters that are read out using electrochemical, gas chromatography, hyperspectral imaging, and next-generation sequencing methods. We also discuss computational advances that are accelerating the diversification of sensing components by mining metagenomics data for new transcriptional regulators and by designing allosteric protein switches that directly regulate reporter outputs using analytes. We highlight emerging opportunities for programming undomesticated microbes in communities to function as distributed sensors in the environment. Finally, we discuss the need for responsible biosensor development and to modernize regulatory frameworks to support evidence-based assessment of environmental biosensors.

analyte↗