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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

Numerical Investigation of a Central Fuel Property Hypothesis under Boosted Spark-ignition Conditions

In the present work, a central fuel property hypothesis (CFPH), which states that fuel properties are sufficient to provide an indication of a fuel's performance irrespective of its chemical composition, was numerically investigated In particular, the objective of the study was to determine whether Research Octane Number (RON) and Motor Octane Number (MON), as fuel properties, are sufficient to describe a fuel's knock-limited performance under boosted spark-ignition (SI) conditions within the framework of CFPH. To this end, four TPRF-bioblendstock surrogates having different compositions but matched RON (= 98) and MON (= 90), were first generated using a non-linear regression model based on artificial neural network (ANN). Three unconventional bioblendstocks were included in the analysis: Di-isobutylene (DIB), Isobutanol and Anisole. Skeletal reaction mechanisms were generated for the TPRF-DIB, TPRF-isobutanol and TPRF-anisole blends from a detailed kinetic mechanism. Thereafter, numerical simulations were performed for the fuel surrogates using the skeletal mechanisms and a virtual cooperative fuel research (CFR) engine model, under a representative boosted operating condition. In the computational fluid dynamics (CFD) model, the G-equation approach was employed to track the turbulent flame front and the well-stirred reactor model combined with multi-zone binning strategy was used to capture auto-ignition in the end-gas. In addition, laminar flame speed was tabulated for each blend as a function of pressure, temperature and equivalence ratio a priori, and the lookup tables were used to prescribe laminar flame speed as an input to the G-equation model. Parametric spark timing sweeps were performed for each fuel blend to determine the corresponding knock-limited spark advance (KLSA) and 50% burn point (CA50) at the respective KLSA timing. It was observed that despite same RON, MON and engine operating conditions, the TPRF-Anisole blend exhibited markedly different knock-limited performance from the other three blends. This deviation from the octane index (DI) expectation was shown to be caused by differences in laminar flame speed (LFS). However; it was found that relatively large fuel-specific differences in LFS (> 20%) would have to be present to cause any appreciable deviation from the 01 framework. Otherwise, RON and MON would still be robust enough to predict a fuel's knock-limited performance.

Pal, Pinaki↗
DOE OSTI2021

Influence of NOx Chemistry on the Prediction of Natural Gas End-Gas Autoignition

Research on the influence of NOx (NO/NO2) chemistry on HC oxidation has shown that NOx plays an important role in the promotion/inhibition of HC autoignition [1-6]. Current data for n-heptane, iso-octane, ethanol, and toluene [3, 4] have shown that NO chemistry inhibits autoignition at low temperatures and concentrations. However, at medium and high temperatures and low concentrations, autoignition is promoted, decreasing the Negative Temperature Coefficient (NTC) effect. This autoignition promotion, however, becomes weaker as NO concentrations are increased. Additionally, fundamental kinetics studies have suggested that NOx chemistry also promotes the autoignition of smaller HC, such as CH4 [7-10], C2H6 [11-14], and C3H8 [15]. Additionally to the chemical kinetics studies, a growing body of evidence supports the importance of NOx chemistry on SI engine simulations [16, 17]. Foong et al. [18] suggested that the addition of NO in their models significantly improved the agreement between simulations and experiments where the onset of knock was advanced due to the presence of NO, and Morganti et al. [19] showed that NO addition in the residual-gas was necessary for their models to obtain good agreement with experimental data. Lastly, Mohr et al. [20, 21] investigated the effect of Natural Gas (NG) reactivity and EGR substitution rate and composition on homogeneous ignition delay, flame speeds, and EGAI for stoichiometric NG/oxidizer/EGR blends. They observed that the addition of EGR composed of only inert species (CO2 and Ar) suppressed homogeneous autoignition in all NG fuels tested under all conditions. However, for all NG fuels tested, increasing EGR rates with reactive species (Ar, CO2, CO, and NO) promoted homogeneous autoignition under all conditions, thus reproducing the same trends for various NG fuel compositions that were observed in other studies and fuels. For this reason, a study is being conducted to analyze the effects of NOx chemistry on the prediction of NG/air/EGR homogeneous autoignition. This study has been divided into two tasks. First, the addition of NOx chemistry to the ARIES82 mechanism [20] is being validated using homogeneous ignition delay data collected by Mohr [20, 21]. Second, once the new modified mechanism with NOx reactions has been validated, it will be employed on engine multi-dimensional modeling presented in [22, 23] to analyze the influence of NOx chemistry on NG EGAI. Results thus far have shown that homogeneous ignition delay calculations are sensitive to NOx chemistry, where the modified mechanisms captured well all trends and closely matched the homogeneous ignition delays observed by Mohr et al. Also, the results in this work suggest that NOx chemistry is necessary to correctly capture the trends in NG homogeneous autoignition when EGR with reactive species is used since the opposite behavior is observed when only inert species are considered for the EGR composition.

03 NATURAL GAS↗
DOE OSTI2019

Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays

Engineering cellular phenotypes often requires the regulation of many genes. When using CRISPR interference, coexpressing many single-guide RNAs (sgRNAs) triggers genetic instability and phenotype loss, due to the presence of repetitive DNA sequences. We stably coexpressed 22 sgRNAs within nonrepetitive extra-long sgRNA arrays (ELSAs) to simultaneously repress up to 13 genes by up to 3,500-fold. We applied biophysical modeling, biochemical characterization and machine learning to develop toolboxes of nonrepetitive genetic parts, including 28 sgRNA handles that bind Cas9. We designed ELSAs by combining nonrepetitive genetic parts according to algorithmic rules quantifying DNA synthesis complexity, sgRNA expression, sgRNA targeting and genetic stability. Using ELSAs, we created three highly selective phenotypes in Escherichia coli, including redirecting metabolism to increase succinic acid production by 150-fold, knocking down amino acid biosynthesis to create a multi-auxotrophic strain and repressing stress responses to reduce persister cell formation by 21-fold. Finally, ELSAs enable simultaneous and stable regulation of many genes for metabolic engineering and synthetic biology applications.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Rewinding evolution in planta: A Rubisco-null platform validates high-performance ancestral enzymes

Improving the photosynthetic enzyme Rubisco is a key target for enhancing C3 crop productivity, but progress has been hampered by the difficulty of evaluating engineered variants in planta without interference from the native enzyme. Here, we report the creation of a Rubisco-null Nicotiana tabacum platform by using CRISPR-Cas9 to knock out all 11 nuclear-encoded small subunit (rbcS) genes. Knockout was achieved in a line expressing cyanobacterial Rubisco from the plastid genome, allowing the recovery of viable plants. We then developed a chloroplast expression system for coexpressing both large and small subunits from the plastid genome. We expressed two resurrected ancestral Rubiscos from the Solanaceae family. The resulting transgenic plants were phenotypically normal and accumulated Rubisco to wild-type levels. Importantly, kinetic analyses of the purified ancestral enzymes revealed they possessed a 16 to 20% higher catalytic efficiency (k cat,air /K c,air ) under ambient conditions, driven by a significantly faster turnover rate (k cat,air ). We have demonstrated that our system allows robust in vivo assessment of novel Rubiscos and that ancestral reconstruction is a powerful strategy for identifying superior enzymes to improve photosynthesis in C3 crops.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Improving CRISPR/Cas9-mediated genome editing efficiency in Yarrowia lipolytica using direct tRNA-sgRNA fusions

We report Yarrowia lipolytica is an important oleaginous yeast currently used in the production of specialty chemicals and has a great potential for further applications in lipid biotechnology. Harnessing the full potential of Y. lipolytica is, however, limited by its inherent recalcitrance to genetic manipulation. In contrast to Saccharomyces cerevisiae, Y. lipolytica is poor in homology-mediated DNA repair and thus in homologous recombination, which limits site-specific gene editing in this yeast. Recently developed CRISPR/Cas9-based methods using tRNA-sgRNA fusions succeeded in editing some genomic loci in Y. lipolytica. Nonetheless, the majority of other tested loci either failed editing or editing was achieved but at very low efficiency using these methods. Using tools of secondary RNA structure prediction, we were able to improve the design of the tRNA-sgRNA fusions used for the expression of single guide RNA (sgRNA) in such methods. This resulted in high efficiency CRISPR/cas9 gene editing at chromosomal loci that failed gene editing or were edited at very low efficiencies with previous methods. In addition, we characterized the gene editing performance of our newly designed tRNA-sgRNA fusions for both chromosomal gene integration and deletion. As such, this study presents an efficient CRISPR/Cas9-mediated gene-editing tool for efficient genetic engineering of Yarrowia lipolytica.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Fuel Stratification Effects on Gasoline Compression Ignition with a Regular-Grade Gasoline on a Single-Cylinder Medium-Duty Diesel Engine at Low Load

Prior research studies have investigated a wide variety of gasoline compression ignition (GCI) injection strategies and the resulting fuel stratification levels to maintain control over the combustion phasing, duration, and heat release rate. Previous GCI research at the US Department of Energy’s Oak Ridge National Laboratory has shown that for a combustion mode with a low degree of fuel stratification, called “partial fuel stratification” (PFS), gasoline range fuels with anti-knock index values in the range of regular-grade gasoline (~87 anti-knock index or higher) provides very little controllability over the timing of combustion without significant boost pressures. On the contrary, heavy fuel stratification (HFS) provides control over combustion phasing but has challenges achieving low temperature combustion operation, which has the benefits of low NOX and soot emissions, because of the air handling burdens associated with the required high exhaust gas recirculation rates. Furthermore, this work investigates HFS and PFS combustion, efficiency, and emissions performance on a single-cylinder, medium-duty engine with a regular-grade gasoline (91 research octane number) at 1,200 rpm, 4.3 bar, and 3.0 nominal gross indicated mean effective pressure operating points with boost levels similar to those in a medium-duty diesel application. Authority of combustion phasing with main injection timing sweeps for HFS and second injection timing sweeps and fuel split sweeps for PFS are shown. In addition, this work is discussed in the context of previous findings with a light-duty diesel platform, and next steps and future direction for this work are presented.

33 ADVANCED PROPULSION SYSTEMS↗
DOE OSTI2020

Programmable gene regulation for metabolic engineering using decoy transcription factor binding sites

Abstract Transcription factor decoy binding sites are short DNA sequences that can titrate a transcription factor away from its natural binding site, therefore regulating gene expression. In this study, we harness synthetic transcription factor decoy systems to regulate gene expression for metabolic pathways in Escherichia coli. We show that transcription factor decoys can effectively regulate expression of native and heterologous genes. Tunability of the decoy can be engineered via changes in copy number or modifications to the DNA decoy site sequence. Using arginine biosynthesis as a showcase, we observed a 16-fold increase in arginine production when we introduced the decoy system to steer metabolic flux towards increased arginine biosynthesis, with negligible growth differences compared to the wild type strain. The decoy-based production strain retains high genetic integrity; in contrast to a gene knock-out approach where mutations were common, we detected no mutations in the production system using the decoy-based strain. We further show that transcription factor decoys are amenable to multiplexed library screening by demonstrating enhanced tolerance to pinene with a combinatorial decoy library. Our study shows that transcription factor decoy binding sites are a powerful and compact tool for metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Uranium Oxide Elutriation at Higher Flow Rates and Longer Times

This work supports the recovery of high-assay low-enriched uranium (i.e., U enriched in 235 U to >5% and <20%) from used nuclear fuel. One step includes elutriating triuranium octoxide (U 3 O 8 ) from a fluidized bed of alumina (Al 2 O 3 ) that fills the main reaction chamber. The experiments outlined in this report assessed the effects of the time and flow rate on the elutriation of U 3 O 8 powder from the reaction vessel. Testing at Oak Ridge National Laboratory (ORNL) was performed in a one-fourth scale experimental apparatus based on a pilot-scale system, which is the Material Recovery Pilot Plant (MRPP) at Idaho National Laboratory (INL). The experimental system comprises a supply of pressurized nitrogen (N 2 ), a gas heater, the reaction vessel, a knock-out pot, and a water trap where elutriated particles are collected for sampling. An Al 2 O 3 fluidized bed is contained within the reaction vessel. A bed of Al 2 O 3 with a nominal size of 180 µm and U 3 O 8 with a nominal size of 7 µm was fluidized at 2 different flow rates so the U 3 O 8 physically separated from the bed. The apparatus was simple and had no valves or sharp bends downstream from the reactor. The flow exited the reactor through a side port at the top. The flow passed through a knock-out vessel before the flow was passed through a bubbler to capture the U 3 O 8 . Seven tests were completed with U 3 O 8 using an elutriating gas of N 2 heated to 200°C. Tests were performed with bed loadings that comprise 25.0 g of U 3 O 8 , or 3.6 wt% of the bed mass. Except for one test performed at 12 SLPM, the gas flow rate through the bed for the other experiments was set at 10 SLPM as measured by the mass flow meter. Linear velocities through the bed were calculated as 28 and 34 cm/s, or 0.9 and 1.1 ft·s -1 . The duration of the tests ranged from 20 to 180 min, and one test had a flow cycled between 0 and 5 SLPM every 5 min for 40 min. The testing successfully demonstrated elutriation of U 3 O 8 from the fluidized bed as up to a 100% of the U 3 O 8 was transferred out of the bed. The removal of U 3 O 8 had a logarithmic dependence on flow rate. The design changes made the apparatus easier to clean, and the mass balances were better than those used in previous studies. Some elutriated Al 2 O 3 collected in the tubing that connected the reactor to the knock-out pot, but the amount of Al 2 O 3 was minimal for 10 SLPM. The flow at 12 SLPM showed considerable transport of Al 2 O 3 bed material into the reaction-vessel headspace and the transfer line to the knock-out pot.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗
DOE OSTI2019

Metabolic engineering of Pseudomonas putida for increased polyhydroxyalkanoate production from lignin

Microbial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic-catabolic pathways are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole-cell biocatalyst for funnelling lignin-derived compounds to value-added products, including its native carbon storage product, medium-chain-length polyhydroxyalkanoates (mcl-PHA). In this work, a series of metabolic engineering targets to improve mcl-PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p-coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene phaZ was knocked out, and the genes involved in β-oxidation (fadBA1 and fadBA2) were deleted. Additionally, to increase carbon flux into mcl-PHA biosynthesis, phaG, alkK, phaC1 and phaC2 were overexpressed. The best performing strain – which contains all the genetic modifications detailed above – demonstrated a 53% and 200% increase in mcl-PHA titre (g l-1) and a 20% and 100% increase in yield (g mcl-PHA per g cell dry weight) from p-coumaric acid and lignin, respectively, compared with the wild type strain. Overall, these results present a promising strain to be employed in further process development for enhancing mcl-PHA production from aromatic compounds and lignin.

09 BIOMASS FUELS↗
DOE OSTI2022

Influence of gasoline fuel formulation on lean autoignition in a mixed-mode-combustion (deflagration/autoignition) engine

We report stoichiometric spark-ignition engines suffer efficiency penalties due to throttling losses at low loads, a low specific-heat ratio of the stoichiometric working fluid, and limits on compression ratio due to end-gas autoignition leading to undesirable knocking. Mixed-Mode Combustion (MMC) mitigates these shortcomings by using a lean working fluid where a spark-initiated pilot-stabilized deflagrative flame front is followed by controlled end-gas autoignition. This MMC study investigates the effects of initial conditions (intake air temperature, intake pressure, equivalence ratio, and intake oxygen fraction) on autoignition tendency of four gasoline-range fuels with varying properties and composition. The use of fuels with varying octane sensitivity (S) allowed exploring the importance of low-temperature heat release in triggering autoignition. Fuels with high S were less reactive for conditions that promote low-temperature chemistry (operation at high intake air pressure or without N2 dilution). Conversely, an Alkylate fuel with low S showed a greater autoignition resistance at operating conditions that were unfavorable for low-temperature chemistry. Next, the effect of residual gas composition on autoignition tendency of fuels was examined with a chemical-kinetics model. Among the various molecules in the residual gas, nitric oxide (NO) enhanced the low-temperature chemistry and increased the autoignition tendency most significantly. The fuels’ autoignition response to increasing NO amount corroborates the experimental observations. Next, the sequential autoignition of the end-gas was assessed to be less impacted by thermal stratification because of lean mixtures showing relatively less low-temperature chemistry, when compared to stoichiometric mixtures. Next, the effect of changing equivalence ratio on the autoignition was found to be similar for all fuels, regardless of their S. With changing intake air temperature, the response of fuels’ autoignition tendency depended on the dilution level used. At high dilution (i.e. low intake [O2]), fuels’ reactivity increased with increasing intake air temperature. In contrast, for operation without dilution, the autoignition tendency of the low-S Alkylate fuel decreased with increasing intake air temperature, while that of high-S High Cycloalkane fuel still increased with increasing intake air temperature. In conclusion, conventional octane metrics (RON and MON) have utility in assessing the autoignition tendency under lean MMC operation. Moreover, the fuel requirements for MMC align with that of stoichiometric operation: i.e., high RON and high S fuels are desirable for stable non-knocking operation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

CRAGE-CRISPR facilitates rapid activation of secondary metabolite biosynthetic gene clusters in bacteria

With the advent of genome sequencing and mining technologies, secondary metabolite biosynthetic gene clusters (BGCs) within bacterial genomes are becoming easier to predict. For subsequent BGC characterization, clustered regularly interspaced short palindromic repeats (CRISPR) has contributed to knocking out target genes and/or modulating their expression; however, CRISPR is limited to strains for which robust genetic tools are available. Here we present a strategy that combines CRISPR with chassis-independent recombinase-assisted genome engineering (CRAGE), which enables CRISPR systems in diverse bacteria. To demonstrate CRAGE-CRISPR, we select 10 polyketide/non-ribosomal peptide BGCs in Photorhabdus luminescens as models and create their deletion and activation mutants. Subsequent loss- and gain-of-function studies confirm 22 secondary metabolites associated with the BGCs, including a metabolite from a previously uncharacterized BGC. These results demonstrate that the CRAGE-CRISPR system is a simple yet powerful approach to rapidly perturb expression of defined BGCs and to profile genotype-phenotype relationships in bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Engineering osmolysis susceptibility in Cupriavidus necator and Escherichia coli for recovery of intracellular products

Intracellular biomacromolecules, such as industrial enzymes and biopolymers, represent an important class of bio-derived products obtained from bacterial hosts. A common key step in the downstream separation of these biomolecules is lysis of the bacterial cell wall to effect release of cytoplasmic contents. Cell lysis is typically achieved either through mechanical disruption or reagent-based methods, which introduce issues of energy demand, material needs, high costs, and scaling problems. Osmolysis, a cell lysis method that relies on hypoosmotic downshock upon resuspension of cells in distilled water, has been applied for bioseparation of intracellular products from extreme halophiles and mammalian cells. However, most industrial bacterial strains are non-halotolerant and relatively resistant to hypoosmotic cell lysis. To overcome this limitation, we developed two strategies to increase the susceptibility of non-halotolerant hosts to osmolysis using Cupriavidus necator, a strain often used in electromicrobial production, as a prototypical strain. In one strategy, C. necator was evolved to increase its halotolerance from 1.5% to 3.25% (w/v) NaCl through adaptive laboratory evolution, and genes potentially responsible for this phenotypic change were identified by whole genome sequencing. The evolved halotolerant strain experienced an osmolytic efficiency of 47% in distilled water following growth in 3% (w/v) NaCl. In a second strategy, the cells were made susceptible to osmolysis by knocking out the large-conductance mechanosensitive channel (mscL) gene in C. necator. When these strategies were combined by knocking out the mscL gene from the evolved halotolerant strain, greater than 90% osmolytic efficiency was observed upon osmotic downshock. A modified version of this strategy was applied to E. coli BL21 by deleting the mscL and mscS (small-conductance mechanosensitive channel) genes. When grown in medium with 4% NaCl and subsequently resuspended in distilled water, this engineered strain experienced 75% cell lysis, although decreases in cell growth rate due to higher salt concentrations were observed. Our strategy is shown to be a simple and effective way to lyse cells for the purification of intracellular biomacromolecules and may be applicable in many bacteria used for bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Bioconversion of Agave Bagasse Lignin to Medium-Chain-Length Polyhydroxyalkanoates by Pseudomonas putida

Inadequate disposal of Agave tequilana bagasse (ATB) brings environmental and economic issues to tequila producing regions. Recent works have proposed technologies for valorization of the polysaccharide’s fractions of ATB. However, lignin bioconversion has not been investigated due to its recalcitrant nature. In this work, a systematic pretreatment is proposed to release the lignocellulosic fractions and produce medium-chain-length polyhydroxyalkanoates (PHA) from the lignin fraction of ATB. Nuclear magnetic resonance (NMR) analyses revealed that ATB lignin contained 63% of β-O-4 interunit linkages, making this lignin more suitable for pretreatment and biodegradation compared to other lignins. Exploratory experiments revealed that two-stage fermentation is suitable for PHA production from ATB lignin using wild type and engineered Pseudomonas putida strains. PHA increased from 0.09 to 0.39 g/L compared to single batch fermentation. Further improvement to 0.76 g/L was possible by using a central composite design to optimize the inoculum, substrate, and nitrogen concentrations. Finally, PHA depolymerase gene phaZ was knocked out from P. putida and tested at optimal conditions, enabling a PHA titer of 0.97 g/L. Analyses by NMR and GC-MS confirmed lignin derivatives consumption and decrease of β-O-4 linkages. This study lays the foundations to enable agave lignin bioprocessing and opens new avenues for ATB biorefineries.

PHA↗
DOE OSTI2020

Self-assembling Shell Proteins PduA and PduJ have Essential and Redundant Roles in Bacterial Microcompartment Assembly

Protein self-assembly is a common and essential biological phenomenon, and bacterial microcompartments present a promising model system to study this process. Bacterial microcompartments are large, protein-based organelles which natively carry out processes important for carbon fixation in cyanobacteria and the survival of enteric bacteria. These structures are increasingly popular with biological engineers due to their potential utility as nanobioreactors or drug delivery vehicles. However, the limited understanding of the assembly mechanism of these bacterial microcompartments hinders efforts to repurpose them for non-native functions. Here, we comprehensively investigate proteins involved in the assembly of the 1,2-propanediol utilization bacterial microcompartment from Salmonella enterica serovar Typhimurium LT2, one of the most widely studied microcompartment systems. We first demonstrate that two shell proteins, PduA and PduJ, have a high propensity for self-assembly upon overexpression, and we provide a novel method for self-assembly quantification. Using genomic knock-outs and knock-ins, we systematically show that these two proteins play an essential and redundant role in bacterial microcompartment assembly that cannot be compensated by other shell proteins. At least one of the two proteins PduA and PduJ must be present for the bacterial microcompartment shell to assemble. We also demonstrate that assembly-deficient variants of these proteins are unable to rescue microcompartment formation, highlighting the importance of this assembly property. Our work provides insight into the assembly mechanism of these bacterial organelles and will aid downstream engineering efforts.

1,2-propanediol utilization MCP↗
DOE OSTI2026

Rational engineering to enhance C8-fatty acid biosynthesis in Picosynechococcus sp. PCC 7002

Medium-chain fatty acids (e.g., C8-C14) are important biofuel precursors that can be produced from CO2 by cyanobacteria and other photoautotrophs. However, cyanobacteria naturally direct a relatively small fraction of fixed carbon to lipid synthesis, primarily producing long-chain membrane-bound fatty acids. We investigated whether mitigating kinetic bottlenecks within the fatty acid biosynthesis (FAB) pathway could enhance flux to free fatty acid (FFA) production in Picosynechococcus sp. PCC 7002. Previous in vitro studies proposed that the FAB initiating enzyme FabH is the primary rate-controlling enzyme in PCC 7002. We hypothesized that enhancing fatty acid initiation could increase in vivo FFA production while shifting the kinetic bottleneck further downstream in the pathway. We enabled C8-FFA accumulation by knocking out the native acyl-acyl carrier protein synthetase gene (aas) and expressing the highly active Cuphea palustris-derived mutant thioesterase CpFatB1.2-M4-287 (CupTE), which selectively catalyzes C8 chain termination. We then expressed the diatom-derived Chaetoceros sp. GSL56 FabH ortholog (chKASIII), to enhance initiation, and a medium-chain selective E. coli ketosynthase ecFabF[I108F] (ecFabF∗), to enhance elongation. When expressed individually or in combination with ecFabF∗, chKASIII slowed growth and decreased net carbon fixation rates relative to the parental Δaas-CupTE strain. However, co-expression of chKASIII, ecFabF∗, and CupTE redirected a larger fraction of fixed carbon toward FFA production, increasing relative carbon flux to C8-FFA and decreasing the projected minimum selling price by four-fold. This work demonstrates how systems metabolic engineering can be applied to enhance C8-FFA production in cyanobacteria while highlighting the unpredictable physiological consequences of host metabolic burden.

09 BIOMASS FUELS↗
DOE OSTI2026

Predictive CRISPR-mediated gene downregulation for enhanced production of sustainable aviation fuel precursor in Pseudomonas putida

CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, its application is often constrained by two challenges: (i) rationally identifying effective gene targets for downregulation and (ii) efficiently constructing multiplexed CRISPRi systems. In this study, we address both challenges by integrating a computational prioritization tool with a versatile assembly method for building multiplexed CRISPRi systems. FluxRETAP (Flux-Reaction Target Prioritization) accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers in Pseudomonas putida KT2440, outperforming a conventional non-computational, pathway-guided target selection. The highest isoprenol titer of nearly 1.5 g/L was achieved by knocking down PP_4118 (a gene encoding α-ketoglutarate dehydrogenase). The use of VAMMPIRE (Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation) enabled accurate assembly of CRISPRi constructs containing up to five sgRNA arrays, reducing context dependency and achieving uniform, position-independent gene downregulation. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.

CRISPR interference↗