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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 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Chapter 12: Gene Editing Technologies for Biofuel Production in Thermophilic Microbes

Thermophilic microbes are an attractive bioproduction platform due to their inherently lower contamination risk and their ability to perform thermostable enzymatic processes which may be required for biomass processing and other industrial applications. The engineering of microbes for industrial scale processes requires a suite of genetic engineering tools to optimize existing biological systems as well as to design and incorporate new metabolic pathways within strains. Yet, such tools are often lacking and/or inadequate for novel microbes, especially thermophiles. This chapter focuses on genetic tool development and engineering strategies, in addition to challenges, for thermophilic microbes. We provide detailed instructions and techniques for tool development for an anaerobic thermophile, Caldanaerobacter subterraneus subsp. tengcongensis, including culturing, plasmid construction, transformation, and selection. This establishes a foundation for advanced genetic tool development necessary for the metabolic engineering of this microbe and potentially other thermophilic organisms.

BASIC BIOLOGICAL SCIENCES,BIOMASS FUELS↗
DOE OSTI2020

Chapter 6: An Improved Leaf Protoplast System for Highly Efficient Transient Expression in Switchgrass (Panicum virgatum L.)

As a robust perennial C4-type monocot plant and a native species to North America, switchgrass (Panicum virgatum) has been evaluated and designated as a strong candidate bioenergy crop by the U.S. DOE. Although genetic modifications of switchgrass have been used to successfully reduce the recalcitrance of switchgrass biomass for biofuel production, the generation of transgenic switchgrass is still a slow and laborious process. A transient protoplast system can provide an excellent platform to accelerate the selection of genes-of-interest for tailoring switchgrass biomass. However, partially due to the lack of the complete genomic information, the attempts to optimize the transient protoplast system for switchgrass remain scarce. In this chapter, we provide an improved protocol for switchgrass protoplast isolation, increased transformation efficiency using CsCl gradient ultracentrifugation-derived plasmid DNA and extended application of the transient switchgrass protoplast system to analyze protein expression using western blot.

BASIC BIOLOGICAL SCIENCES,BIOMASS FUELS↗
DOE OSTI2024

Pore confinement enhances but surface adhesion reduces bacterial cell-to-cell conjugation

As the habitats of bacteria, soil pore network and surface properties control the distribution, adhesion, and motility of bacteria in soils. These physical processes in turn influence bacterial accesses to nutrients and bacterial interactions. Our understanding on the pore- and surface-mediated bacterial interactions is currently limited. In this research, we evaluated the effects of soil pore confinement and surface adhesion on conjugation-based bacterial interactions. The interaction was measured by plasmid transfer between donor and recipient cells within the population of soil bacterium Pseudomonas putida. We found that the presence of porous sand media led to a net increase in conjugation frequency compared to sand-free liquid control. The increase is attributed to the facilitated effect of pore confinement on the collision of bacteria within pores. In contrast, bacterial adhesion to sand surfaces under elevated ionic strength conditions decreased the conjugation frequency as a result of mobility reduction on the surface. Such collision and adhesion mechanisms jointly drive the conjugation as a function of pore and surface properties of porous media. Finally, these results provide valuable insights into the roles of soil pores and surfaces in regulating horizontal gene transfer, an essential cell-to-cell interaction sustaining key processes of soil ecology and health.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Deletion of the cytochrome bc complex from Heliobacterium modesticaldum results in viable but non-phototrophic cells

The heliobacteria, a family of anoxygenic phototrophs, possess the simplest known photosynthetic apparatus. Although they are photoheterotrophs in the light, the heliobacteria can also grow chemotrophically via pyruvate metabolism in the dark. In the heliobacteria, the cytochrome bc complex is responsible for oxidizing menaquinol and reducing cytochrome c 553 in the electron flow cycle used for phototrophy. However, there is no known electron acceptor for the mobile cytochrome c 553 other than the photochemical reaction center. We have, therefore, hypothesized that the cytochrome bc complex is necessary for phototrophy, but unnecessary for chemotrophic growth in the dark. Here, we used a two-step method for CRISPR-based genome editing in Heliobacterium modesticaldum to delete the genes encoding the four major subunits of the cytochrome bc complex. Genotypic analysis verified the deletion of the petCBDA gene cluster encoding the catalytic components of the complex. Spectroscopic studies revealed that re-reduction of cytochrome c 553 after flash-induced photo-oxidation was over 100 times slower in the petCBDA mutant compared to the wild-type. Steady-state levels of oxidized P 800 (the primary donor of the photochemical reaction center) were much higher in the petCBDA mutant at every light level, consistent with a limitation in electron flow to the reaction center. The petCBDA mutant was unable to grow phototrophically on acetate plus CO 2 but could grow chemotrophically on pyruvate as a carbon source similar to the wild-type strain in the dark. The mutants could be complemented by reintroduction of the petCBDA gene cluster on a plasmid expressed from the clostridial eno promoter.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The PshX subunit of the photochemical reaction center from Heliobacterium modesticaldum acts as a low-energy antenna

The anoxygenic phototrophic bacterium Heliobacterium modesticaldum contains a photochemical reaction center protein complex (called the HbRC) consisting of a homodimer of the PshA polypeptide and two copies of a newly discovered polypeptide called PshX, which is a single transmembrane helix that binds two bacteriochlorophyll g molecules. Here, to assess the function of PshX, we produced a ΔpshX strain of Hbt. modesticaldum by leveraging the endogenous Hbt. modesticaldum Type I-A CRISPR-Cas system to aid in mutant selection. We optimized this system by separating the homologous recombination and CRISPR-based selection steps into two plasmid transformations, allowing for markerless gene replacement. Fluorescence and low-temperature absorbance of the purified HbRC from the wild-type and ΔpshX strains showed that the bacteriochlorophylls bound by PshX have the lowest site energies in the entire HbRC. This indicates that PshX acts as a low-energy antenna subunit, participating in entropy-assisted uphill energy transfer towards the P 800 special bacteriochlorophyll g pair. We further discuss the role that PshX may play in stability of the HbRC, its conservation in other heliobacterial species, and the evolutionary pressure to produce and maintain single-TMH subunits in similar locations in other reaction centers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Photosynthesis | The Heliobacteria

The heliobacteria are the only phototrophs in the Gram-positive group of bacteria. They are strict anaerobes that grow in a primarily fermentative mode in the dark. In the light, they use a homodimeric Type I photochemical reaction center to drive cyclic electron transport. They are unique in using the pigment bacteriochlorophyll g. All heliobacteria are unable to fix CO 2 but able to fix N2. Their core metabolism includes a complete TCA cycle with several reversible enzymes in common with the reverse (reductive) TCA cycle, but they lack an (ATP) citrate lyase that would allow CO 2 fixation. There are now several sequenced genomes available for different heliobacterial species, allowing hypotheses to be made concerning their metabolic potential. Here, techniques to introduce replicating plasmids into Heliobacterium modesticaldum, as well as leveraging its endogenous CRISPR/Cas system to edit its chromosome, have recently been pioneered, contributing to making this species a model system for this family.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Human DNA polymerase η promotes RNA-templated error-free repair of DNA double-strand breaks

A growing body of evidence indicates that RNA plays a critical role in orchestrating DNA double-strand break repair (DSBR). Recently, we showed that homologous nascent RNA can be used as a template for error-free repair of double-strand breaks (DSBs) in the transcribed genome and to restore the missing sequence at the break site via the transcription-coupled classical nonhomologous end-joining (TC-NHEJ) pathway. TC-NHEJ is a complex multistep process in which a reverse transcriptase (RT) is essential for synthesizing the DNA strand from template RNA. However, the identity of the RT involved in the TC-NHEJ pathway remained unknown. Here, we report that DNA polymerase eta (Pol η), known to possess RT activity, plays a critical role in TC-NHEJ. We found that Pol η forms a multiprotein complex with RNAP II and other TC-NHEJ factors, while also associating with nascent RNA. Moreover, purified Pol η, along with DSBR proteins PNKP, XRCC4, and Ligase IV can fully repair RNA templated 3'-phosphate-containing gapped DNA substrate. In addition, we demonstrate here that Pol η deficiency leads to accumulation of R-loops and persistent strand breaks in the transcribed genes. Finally, we determined that, in Pol η depleted but not in control cells, TC-NHEJ-mediated repair was severely abrogated when a reporter plasmid containing a DSB with several nucleotide deletion within the E. coli lacZ gene was introduced for repair in lacZ-expressing mammalian cells. Thus, our data strongly suggest that RT activity of Pol η is required in error-free DSBR.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Engineering Citrobacter freundii using CRISPR/Cas9 system

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins) system is a useful tool to edit genomes quickly and efficiently. However, the use of CRISPR/Cas9 to edit bacterial genomes has been limited to select microbial chassis primarily used for bioproduction of high value products. Thus, expansion of CRISPR/Cas9 tools to other microbial organisms is needed. Here, our aim was to assess the suitability of CRISPR/Cas9 for genome editing of the Citrobacter freundii type strain ATCC 8090. We evaluated the commonly used two plasmid pCas/pTargetF system to enable gene deletions and insertions in C. freundii and determined editing efficiency. The CRISPR/Cas9 based method enabled high editing efficiency (~91%) for deletion of galactokinase (galk) and enabled deletion with various single guide RNA (sgRNA) sequences. To assess the ability of CRISPR/Cas9 tools to insert genes, we used the fluorescent reporter mNeonGreen, an endopeptidase (yebA), and a transcriptional regulator (xylS) and found successful insertion with high efficiency (81-100%) of each gene individually. These results strengthen and expand the use of CRISPR/Cas9 genome editing to C. freundii as an additional microbial chassis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Expanding the genomic encyclopedia of Actinobacteria with 824 isolate reference genomes

The phylum Actinobacteria includes important human pathogens like Mycobacterium tuberculosis and Corynebacterium diphtheriae and renowned producers of secondary metabolites of commercial interest, yet only a small part of its diversity is represented by sequenced genomes. Here, we present 824 actinobacterial isolate genomes in the context of a phylum-wide analysis of 6,700 genomes including public isolates and metagenome-assembled genomes (MAGs). We estimate that only 30%–50% of projected actinobacterial phylogenetic diversity possesses genomic representation via isolates and MAGs. A comparison of gene functions reveals novel determinants of host-microbe interaction as well as environment-specific adaptations such as potential antimicrobial peptides. We identify plasmids and prophages across isolates and uncover extensive prophage diversity structured mainly by host taxonomy. Analysis of >80,000 biosynthetic gene clusters reveals that horizontal gene transfer and gene loss shape secondary metabolite repertoire across taxa. Our observations illustrate the essential role of and need for high-quality isolate genome sequences.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Controlling selectivity of modular microbial biosynthesis of butyryl-CoA-derived designer esters

Short-chain esters have broad utility as flavors, fragrances, solvents, and biofuels. Controlling selectivity of ester microbial biosynthesis has been an outstanding metabolic engineering problem. In this study, we enabled the de novo fermentative microbial biosynthesis of butyryl-CoA-derived designer esters (e.g., butyl acetate, ethyl butyrate, butyl butyrate) in Escherichia coli with controllable selectivity. Using the modular design principles, we generated the butyryl-CoA-derived ester pathways as exchangeable production modules compatible with an engineered chassis cell for anaerobic production of designer esters. We designed these modules derived from an acyl-CoA submodule (e.g., acetyl-CoA, butyryl-CoA), an alcohol submodule (e.g., ethanol, butanol), a cofactor regeneration submodule (e.g., NADH), and an alcohol acetyltransferase (AAT) submodule (e.g., ATF1, SAAT) for rapid module construction and optimization by manipulating replication (e.g., plasmid copy number), transcription (e.g., promoters), translation (e.g., codon optimization), pathway enzymes, and pathway induction conditions. To further enhance production of designer esters with high selectivity, we systematically screened various strategies of protein solubilization using protein fusion tags and chaperones to improve the soluble expression of multiple pathway enzymes. Finally, our engineered ester-producing strains could achieve 19-fold increase in butyl acetate production (0.64 g/L, 96% selectivity), 6-fold increase in ethyl butyrate production (0.41 g/L, 86% selectivity), and 13-fold increase in butyl butyrate production (0.45 g/L, 54% selectivity) as compared to the initial strains. Altogether, this study presented a generalizable framework to engineer modular microbial platforms for anaerobic production of butyryl-CoA-derived designer esters from renewable feedstocks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Corrigendum to "Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440" [Metab. Eng. 59 (2020) 64-75]

The plasmid used to delete hexR, pGB022, in our Pseudomonas putida KT2440 strain engineered to produce cis,cis-muconic acid from glucose, GB062, inadvertently introduced two mutations: a point mutation in the zwf-1, encoding the glucose-6-phosphate 1-dehydrogenase, that results in a conservative, V157I replacement and a deletion of the last 8 bp of the glucose-6-phosphate 1-epimerase gene, yeaD, leading to a frame shift in this gene and, consequently, the replacement of the last two amino acids of the C-terminus (LS) with 56 amino acids (RGPWVCPWVGKTAGCVSSRLFARSSIVLSVRLTYHLSVTLVCNVVVFTTLSLENRV).

BIOMASS FUELS↗
DOE OSTI2023

A landing pad system for multicopy gene integration in Issatchenkia orientalis

The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user’s choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. Furthermore, this study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Development of Corynebacterium glutamicum as a monoterpene production platform

Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at 1504.6 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Metabolic flux optimization of iterative pathways through orthogonal gene expression control: Application to the β-oxidation reversal

Balancing relative expression of pathway genes to minimize flux bottlenecks and metabolic burden is one of the key challenges in metabolic engineering. This is especially relevant for iterative pathways, such as reverse β-oxidation (rBOX) pathway, which require control of flux partition at multiple nodes to achieve efficient synthesis of target products. Here, we develop a plasmid-based inducible system for orthogonal control of gene expression (referred to as the TriO system) and demonstrate its utility in the rBOX pathway. Leveraging effortless construction of TriO vectors in a plug-and-play manner, we simultaneously explored the solution space for enzyme choice and relative expression levels. Remarkably, varying individual expression levels led to substantial change in product specificity ranging from no production to optimal performance of about 90% of the theoretical yield of the desired products. We obtained titers of 6.3 g/L butyrate, 2.2 g/L butanol and 4.0 g/L hexanoate from glycerol in E. coli, which exceed the best titers previously reported using equivalent enzyme combinations. Since a similar system behavior was observed with alternative termination routes and higher-order iterations, we envision our approach to be broadly applicable to other iterative pathways besides the rBOX. Here, considering that high throughput, automated strain construction using combinatorial promoter and RBS libraries remain out of reach for many researchers, especially in academia, tools like the TriO system could democratize the testing and evaluation of pathway designs by reducing cost, time and infrastructure requirements.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

High-Throughput Microfluidics Platform for Intracellular Delivery and Sampling of Biomolecules from Live Cells

Nondestructive cell membrane permeabilization systems enable the intracellular delivery of exogenous biomolecules for cell engineering tasks as well as the temporal sampling of cytosolic contents from live cells for the analysis of dynamic processes. Here, in this paper, we report a microwell array format live-cell analysis device (LCAD) that can perform localized-electroporation induced membrane permeabilization, for cellular delivery or sampling, and directly interfaces with surface-based biosensors for analyzing the extracted contents. We demonstrate the capabilities of the LCAD via an automated high-throughput workflow for multimodal analysis of live-cell dynamics, consisting of quantitative measurements of enzyme activity using self-assembled monolayers for MALDI mass spectrometry (SAMDI) and deep-learning enhanced imaging and analysis. By combining a fabrication protocol that enables robust assembly and operation of multilayer devices with embedded gold electrodes and an automated imaging workflow, we successfully deliver functional molecules (plasmid and siRNA) into live cells at multiple time-points and track their effect on gene expression and cell morphology temporally. Furthermore, we report sampling performance enhancements, achieving saturation levels of protein tyrosine phosphatase activity measured from as few as 60 cells, and demonstrate control over the amount of sampled contents by optimization of electroporation parameters using a lumped model. Lastly, we investigate the implications of cell morphology on electroporation-induced sampling of fluorescent molecules using a deep-learning enhanced image analysis workflow.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

An Improved CRISPR Interference Tool to Engineer Rhodococcus opacus

Rhodococcus opacus is a non-model bacterium that is well suited for valorizing lignin. Despite recent advances in our systems-level understanding of its versatile metabolism, studies of its gene functions at a single gene level are still lagging. Elucidating gene functions in non-model organisms is challenging due to limited genetic engineering tools that are convenient to use. To address this issue, we developed a simple gene repression system based on CRISPR interference (CRISPRi). This gene repression system uses a T 7 RNA polymerase system to express a small guide RNA, demonstrating improved repression compared to the previously demonstrated CRISPRi system (i.e., the maximum repression efficiency improved from 58% to 85%). Additionally, our cloning strategy allows for building multiple CRISPRi plasmids in parallel without any PCR step, facilitating the engineering of this GC-rich organism. Using the improved CRISPRi system, we confirmed the annotated roles of four metabolic pathway genes, which had been identified by our previous transcriptomic analysis to be related to the consumption of benzoate, vanillate, catechol, and acetate. Furthermore, we showed our tool’s utility by demonstrating the inducible accumulation of muconate that is a precursor of adipic acid, an important monomer for nylon production. While the maximum muconate yield obtained using our tool was 30% of the yield obtained using gene knockout, our tool showed its inducibility and partial repressibility. In conclusion, our CRISPRi tool will be useful to facilitate functional studies of this non-model organism and engineer this promising microbial chassis for lignin valorization.

09 BIOMASS FUELS↗
DOE OSTI2022

Crowding-Induced Spatial Organization of Gene Expression in Cell-Sized Vesicles

Cell-free protein synthesis is an important tool for studying gene expression and harnessing it for applications. In cells, gene expression is regulated in part by the spatial organization of transcription and translation. Unfortunately, current cell-free approaches are unable to control the organization of molecular components needed for gene expression, which limits the ability to probe and utilize its effects. In this work, we show, using complementary computational and experimental approaches, that macromolecular crowding can be used to control the spatial organization and translational efficiency of gene expression in cell-sized vesicles. Computer simulations and imaging experiments reveal that, as crowding is increased, DNA plasmids become localized at the inner surface of vesicles. Ribosomes, in contrast, remain uniformly distributed, demonstrating that crowding can be used to differentially organize components of gene expression. We further carried out cell-free protein synthesis reactions in cell-sized vesicles and quantified mRNA and protein abundance. At sufficiently high levels of crowding, we observed localization of mRNA near vesicle surfaces, a decrease in translational efficiency and protein abundance, and anomalous scaling of protein abundance as a function of vesicle size. These results are consistent with high levels of crowding causing altered spatial organization and slower diffusion. Our work demonstrates a straightforward way to control the organization of gene expression in cell-sized vesicles and provides insight into the spatial regulation of gene expression in cells.

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

A Suite of Constitutive Promoters for Tuning Gene Expression in Plants

The need for convenient tools to express transgenes over a large dynamic range is pervasive throughout plant synthetic biology; however, current efforts are largely limited by the heavy reliance on a small set of strong promoters, precluding more nuanced and refined engineering endeavors in planta . To address this technical gap, we characterize a suite of constitutive promoters that span a wide range of transcriptional levels and develop a GoldenGate-based plasmid toolkit named PCONS, optimized for versatile cloning and rapid testing of transgene expression at varying strengths. We demonstrate how easy access to a stepwise gradient of expression levels can be used for optimizing synthetic transcriptional systems and the production of small molecules in planta . We also systematically investigate the potential of using PCONS as an internal standard in plant biology experimental design, establishing the best practices for signal normalization in experiments. Although our library has primarily been developed for optimizing expression in N. benthamiana , we demonstrate the translatability of our promoters across distantly related species using a multiplexed reporter assay with barcoded transcripts. In conclusion, our findings showcase the advantages of the PCONS library as an invaluable toolkit for plant synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗