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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 235 records · Page 13
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

The ‘photosynthetic C 1 pathway’ links carbon assimilation and growth in California poplar

Although primarily studied in relation to photorespiration, serine metabolism in chloroplasts may play a key role in plant CO 2 fertilization responses by linking CO 2 assimilation with growth. Here, we show that the phosphorylated serine pathway is part of a 'photosynthetic C 1 pathway' and demonstrate its high activity in foliage of a C 3 tree where it rapidly integrates photosynthesis and C 1 metabolism contributing to new biomass via methyl transfer reactions, imparting a large natural 13 C-depleted signature. Using 13 CO 2 -labelling, we show that leaf serine, the S-methyl group of leaf methionine, pectin methyl esters, and the associated methanol released during cell wall expansion during growth, are directly produced from photosynthetically-linked C 1 metabolism, within minutes of light exposure. We speculate that the photosynthetic C 1 pathway is highly conserved across the photosynthetic tree of life, is responsible for synthesis of the greenhouse gas methane, and may have evolved with oxygenic photosynthesis by providing a mechanism of directly linking carbon and ammonia assimilation with growth. Although the rise in atmospheric CO 2 inhibits major metabolic pathways like photorespiration, our results suggest that the photosynthetic C 1 pathway may accelerate and represents a missing link between enhanced photosynthesis and plant growth rates during CO 2 fertilization under a changing climate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A comprehensive strategy for stable, high productivity cultivation of microalgae with controllable biomass composition

This project was performed primarily as a partnership between three Universities – The University of Toledo (UToledo), Montana State University (MSU) and The University of North Carolina at Chapel Hill (UNC). North Carolina State University also contributed to the project through a sub-subcontract. The project developed fundamental and applied knowhow for cultivation systems that are not restricted by (i) proximate availability of flue gas or other high concentration CO 2 sources and (ii) the energy and infrastructure burden to deliver CO 2 over long distances. To overcome these constraints, this project focused on cultivation of alkaliphilic algal cultures in high pH, high alkalinity media (pH>10, alkalinity >100mEq) and demonstrated high biomass productivity, improvement of biomass composition for fuel production, and culture stability. The high media alkalinity ensured availability of sufficient HCO 3 - in solution for high rates of carbon fixation while the high pH of the media facilitated rapid scavenging of atmospheric CO 2 . Overall, the direct air capture rates (DAC) in our system were sufficient to achieve high biomass productivities using atmospheric CO 2 alone. The project also developed and validated toolkits related to metabolic flux modeling and microbiome characterization.

09 BIOMASS FUELS↗
DOE OSTI2024

Seasonal Controls on Microbial Depolymerization and Oxidation of Organic Matter in Floodplain Soils

Floodplain soils are vast reservoirs of organic carbon often attributed to anaerobic conditions that impose metabolic constraints on organic matter degradation. What remains elusive is how such metabolic constraints respond to dynamic flooding and drainage cycles characteristic of floodplain soils. Here, in this work, we show that microbial depolymerization and respiration of organic compounds, two rate-limiting steps in decomposition, vary spatially and temporally with seasonal flooding of mountainous floodplain soils (Gothic, Colorado, USA). Combining metabolomics and -proteomics, we found a lower abundance of oxidative enzymes during flooding coincided with the accumulation of aromatic, high-molecular weight compounds, particularly in surface soils. In subsurface soils, we found that a lower oxidation state of carbon coincided with a greater abundance of chemically reduced, energetically less favorable low-molecular weight metabolites, irrespective of flooding condition. Our results suggest that seasonal flooding temporarily constrains oxidative depolymerization of larger, potentially plant-derived compounds in surface soils; in contrast, energetic constraints on microbial respiration persist in more reducing subsurface soils regardless of flooding. Our work underscores that the potential vulnerability of these distinct anaerobic carbon storage mechanisms to changing flooding dynamics should be considered, particularly as climate change shifts both the frequency and extent of flooding in floodplains globally.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Nitrogen Source Governs Community Carbon Metabolism in a Model Hypersaline Benthic Phototrophic Biofilm

Increasing anthropogenic inputs of fixed nitrogen are leading to greater eutrophication of aquatic environments, but it is unclear how this impacts the flux and fate of carbon in lacustrine and riverine systems. Here, we present evidence that the form of nitrogen governs the partitioning of carbon among members in a genome-sequenced, model phototrophic biofilm of 20 members. Consumption of NO 3 – as the sole nitrogen source unexpectedly resulted in more rapid transfer of carbon to heterotrophs than when NH 4 + was also provided, suggesting alterations in the form of carbon exchanged. The form of nitrogen dramatically impacted net community nitrogen, but not carbon, uptake rates. Furthermore, this alteration in nitrogen form caused very large but focused alterations to community structure, strongly impacting the abundance of only two species within the biofilm and modestly impacting a third member species. Our data suggest that nitrogen metabolism may coordinate coupled carbon-nitrogen biogeochemical cycling in benthic biofilms and, potentially, in phototroph-heterotroph consortia more broadly. It further indicates that the form of nitrogen inputs may significantly impact the contribution of these communities to carbon partitioning across the terrestrial-aquatic interface.

carbon cycling↗
DOE OSTI2020

Lignin lags, leads, or limits the decomposition of litter and soil organic carbon

Lignin’s role in litter and soil organic carbon (SOC) decomposition remains contentious. Lignin decomposition was traditionally thought to increase during midstage litter decomposition, when cellulose occlusion by lignin began to limit mass loss. Alternatively, lignin decomposition could be greatest in fresh litter as a consequence of co-metabolism, and lignin might decompose faster than bulk SOC. To test these competing hypotheses, we incubated 10 forest soils with C 4 grass litter (amended with 13 C-labeled or unlabeled lignin) over 2 yr and measured soil respiration and its isotope composition. Early lignin decomposition was greatest in 5 of 10 soils, consistent with the co-metabolism hypothesis. However, lignin decomposition peaked 6–24 months later in the other five soils, consistent with the substrate-limitation hypothesis; these soils were highly acidic. Rates of lignin, litter, and SOC decomposition tended to converge over time. Cumulative lignin decomposition was never greater than SOC decomposition; lignin decomposition was significantly lower than SOC decomposition in six soils. Net nitrogen mineralization predicted lignin decomposition ratios relative to litter and SOC. Although the onset of lignin decomposition can indeed be rapid, lignin still presents a likely bottleneck in litter and SOC decomposition, meriting a reconsideration of lignin’s role in modern decomposition paradigms.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Whole-plant phenotypic engineering: moving beyond ratios for multi-objective optimization of nutrient use efficiency

Nutrient use efficiency (NUE) is typically measured as the ratio of yield to soil nutrient availability but ignores contributions of underlying plant traits. Relevant plant traits can be grouped as root acquisition efficiency, shoot radiation use efficiency, and plant metabolic efficiency. The intentional integration of these traits will lead to synergistic improvements of NUE. Recent progress in trait-focused research includes phenotyping root nutrient uptake rates and respiration, engineering reduced photorespiration, and identification of nutrient assimilation pathways. Traits need to be conceptualized in agricultural systems contexts to improve synchrony of plant demand and soil supply of nutrients, including consideration of crop mixtures. Use of simulation modeling and multi-objective optimization will allow accelerating NUE gains beyond selection for a single ratio.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Versatile microbial communities rapidly assimilate ammonium hydroxide-treated plastic waste

Abstract Waste plastic presently accumulates in landfills or the environment. While natural microbial metabolisms can degrade plastic polymers, biodegradation of plastic is very slow. This study demonstrates that chemical deconstruction of polyethylene terephthalate (PET) with ammonium hydroxide can replace the rate limiting step (depolymerization) and by producing plastic-derived terephthalic acid and terephthalic acid monoamide. The deconstructed PET (DCPET) is neutralized with phosphoric acid prior to bioprocessing, resulting in a product containing biologically accessible nitrogen and phosphorus from the process reactants. Three microbial consortia obtained from compost and sediment degraded DCPET in ultrapure water and scavenged river water without addition of nutrients. No statistically significant difference was observed in growth rate compared to communities grown on DCPET in minimal culture medium. The consortia were dominated by Rhodococcus spp., Hydrogenophaga spp., and many lower abundance genera. All taxa were related to species known to degrade aromatic compounds. Microbial consortia are known to confer flexibility in processing diverse substrates. To highlight this, we also demonstrate that two microbial consortia can grow on similarly deconstructed polyesters, polyamides, and polyurethanes in water instead of medium. Our findings suggest that microbial communities may enable flexible bioprocessing of mixed plastic wastes when coupled with chemical deconstruction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Generating Co-expression Networks for Three Cyanobacteria: Synechococcus sp. PCC 7942, Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803

Cyanobacteria are photosynthetic organisms capable of high growth rate and represent a promising bioplatform for harnessing the sun’s energy to make biofuel. Additionally, the process of photosynthesis absorbs CO2 from the environment. Understanding the metabolic processes involved in photosynthesis could lead to solutions to the recent rise of CO2 concentration in Earth’s atmosphere and the associated climate change. More research on the transcriptional regulation of these cells is needed to learn how to harness the untapped potential of cyanobacteria for these applications. Transcriptional analysis via RNA-seq provides an understanding of how gene expression changes at the mRNA level under diverse growing conditions. I systematically collected and analyzed RNA-Seq data obtained under a variety of conditions and available on the NCBI database for three cyanobacteria model organisms: Synechococcus elongatus sp. PCC 7942, Synechococcus sp. PCC 7002, and Synechocystis sp. PCC 6803. For each organism, the data was mapped to a reference genome to characterize the RNA expression profile. Samples were checked for quality based on the number of reads and the correlation of the expression profile between labeled replicates. All samples were transformed into transcripts per million reads, followed by a log transformation to account for the wide range of sample sizes. Gene co-expression networks were generated and analyzed for each species using Cytoscape. These networks provide a base level of gene expression for each species. The network topology and high-betweeness nodes of these networks need to be analyzed further to provide insight on potential ways to harness cyanobacteria genetics. Additionally, these datasets can be used together to form a core genome network analysis- one that includes only the genes that are homologous between the three species. This project has prepared the way for a more in-depth study on photosynthetic microbes on a genetic level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Computational design and engineering of an Escherichia coli strain producing the nonstandard amino acid para -aminophenylalanine

Introducing heterologous pathways into host cells constitutes a promising strategy for synthesizing nonstandard amino acids (nsAAs) to enable the production of proteins with expanded chemistries. However, this strategy has proven challenging, as the expression of heterologous pathways can disrupt cellular homeostasis of the host cell. Here, we sought to optimize the heterologous production of the nsAA para -aminophenylalanine (pAF) in Escherichia coli . First, we incorporated a heterologous pAF biosynthesis pathway into a genome-scale model of E. coli metabolism and computationally identified metabolic interventions in the host’s native metabolism to improve pAF production. Next, we explored different approaches of imposing these flux interventions experimentally and found that the upregulation of flux in the chorismate biosynthesis pathway through the elimination of feedback inhibition mechanisms could significantly raise pAF titers (~20-fold) while maintaining a reasonable pAF production-growth rate trade-off. Overall, this study provides a promising strategy for the biosynthesis of nsAAs in engineered cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Adaptive laboratory evolution and genetic engineering improved terephthalate utilization in Pseudomonas putida KT2440

Poly(ethylene terephthalate) (PET) is one of the most ubiquitous plastics and can be depolymerized through biological and chemo-catalytic routes to its constituent monomers, terephthalic acid (TPA) and ethylene glycol (EG). TPA and EG can be re-synthesized into PET for closed-loop recycling or microbially converted into higher-value products for open-loop recycling. Here, in this study, we expand on our previous efforts engineering and applying Pseudomonas putida KT2440 for PET conversion by employing adaptive laboratory evolution (ALE) to improve TPA catabolism. Three P. putida strains with varying degrees of metabolic engineering for EG catabolism underwent an automation-enabled ALE campaign on TPA, a TPA and EG mixture, and glucose as a control. ALE increased the growth rate on TPA and TPA-EG mixtures by 4.1- and 3.5-fold, respectively, in approximately 350 generations. Evolved isolates were collected at the midpoints and endpoints of 39 independent ALE experiments, and growth rates were increased by 0.15 and 0.20 h -1 on TPA and a TPA-EG, respectively, in the best performing isolates. Whole-genome re-sequencing identified multiple converged mutations, including loss-of-function mutations to global regulators gacS, gacA, and turA along with large duplication and intergenic deletion events that impacted the heterologously-expressed tphAB II catabolic genes. Reverse engineering of these targets confirmed causality, and a strain with all three regulators deleted and second copies of tphAB II and tpaK displayed improved TPA utilization compared to the base strain. Taken together, an iterative strain engineering process involving heterologous pathway engineering, ALE, whole genome sequencing, and genome editing identified five genetic interventions that improve P. putida growth on TPA, aimed at developing enhanced whole-cell biocatalysts for PET upcycling.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Metabolic Engineering of Nonmodel Yeast Issatchenkia orientalis SD108 for 5–Aminolevulinic Acid Production

Biological production of 5-aminolevulinic acid (5-ALA) has received growing attention over the years. However, there is the tradeoff between 5-ALA biosynthesis and cell growth because the fermentation broth will become acidic due to the production of 5-ALA. To address this limitation, we engineered an acid-tolerant yeast, Issatchenkia orientalis SD108, for 5-ALA production. We first discovered that the cell growth rate of I. orientalis SD108 was boosted by 5-ALA and its endogenous ALA synthetase (ALAS) showed higher activity than those homologs from other yeasts. The titer of 5-ALA was improved from 28 mg/L to 120-, 150-, and 300 mg/L, by optimizing plasmid design, overexpressing a transporter, and increasing gene copy number, respectively. After redirecting the metabolic flux using the pyruvate decarboxylase (PDC) knockout strain (SD108ΔPDC) and culturing with urea, we increased the titer of 5-ALA to 510 mg/L, a 13-fold enhancement, proving the importance of the newly identified IoALAS with higher activity and the strategic selection of nitrogen sources for knockout strains. This study demonstrates the acid-tolerant I. orientalis SD108ΔPDC has a high potential for 5-ALA production at a large scale in the future.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Gene targets for engineering osmotolerance in Caldicellulosiruptor bescii

Abstract Background Caldicellulosiruptor bescii , a promising biocatalyst being developed for use in consolidated bioprocessing of lignocellulosic materials to ethanol, grows poorly and has reduced conversion at elevated medium osmolarities. Increasing tolerance to elevated fermentation osmolarities is desired to enable performance necessary of a consolidated bioprocessing (CBP) biocatalyst. Results Two strains of C. bescii showing growth phenotypes in elevated osmolarity conditions were identified. The first strain, ORCB001, carried a deletion of the FapR fatty acid biosynthesis and malonyl-CoA metabolism repressor and had a severe growth defect when grown in high-osmolarity conditions—introduced as the addition of either ethanol, NaCl, glycerol, or glucose to growth media. The second strain, ORCB002, displayed a growth rate over three times higher than its genetic parent when grown in high-osmolarity medium. Unexpectedly, a genetic complement ORCB002 exhibited improved growth, failing to revert the observed phenotype, and suggesting that mutations other than the deleted transcription factor (the fruR/cra gene) are responsible for the growth phenotype observed in ORCB002. Genome resequencing identified several other genomic alterations (three deleted regions, three substitution mutations, one silent mutation, and one frameshift mutation), which may be responsible for the observed increase in osmolarity tolerance in the fruR / cra -deficient strain, including a substitution mutation in dnaK , a gene previously implicated in osmoresistance in bacteria. Differential expression analysis and transcription factor binding site inference indicates that FapR negatively regulates malonyl-CoA and fatty acid biosynthesis, as it does in many other bacteria. FruR/Cra regulates neighboring fructose metabolism genes, as well as other genes in global manner. Conclusions Two systems able to effect tolerance to elevated osmolarities in C. bescii are identified. The first is fatty acid biosynthesis. The other is likely the result of one or more unintended, secondary mutations present in another transcription factor deletion strain. Though the locus/loci and mechanism(s) responsible remain unknown, candidate mutations are identified, including a mutation in the dnaK chaperone coding sequence. These results illustrate both the promise of targeted regulatory manipulation for osmotolerance (in the case of fapR ) and the challenges (in the case of fruR/cra ).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Effects of Sodium and Magnesium Ions on the Photochemically Induced Heterogeneous Formation of Manganese Oxides and Their Structural Evolution

Manganese (Mn) oxides are abundant in aquatic and terrestrial environments, where they play significant roles in redox cycling and biological metabolisms. We recently observed that Mn oxides were homogeneously formed during the abiotic oxidation of Mn 2+ (aq) to Mn(IV) by O 2 •– via nitrate photolysis, at a rate comparable to that of biotic Mn oxide formation. On the other hand, for the heterogeneous formation of Mn oxides, the presence of a substrate can alter the required thermodynamic driving force, which may affect their crystalline phases and further influence the oxidative capability of redox cycling in environmental systems. However, little is known about the photochemically induced heterogeneous formation of Mn oxides on substrates. In this study, we investigated the heterogeneous formation of Mn oxides on a quartz substrate in the presence of two environmentally abundant cations, Na + and Mg 2+ . In contrast to homogeneously generated Mn oxides, the heterogeneously formed Mn oxides displayed faster crystalline phase evolutions and morphological changes over time. Additionally, the coexistence of Na + and Mg 2+ ions greatly affected the initial crystalline phases and the phase evolution, as well as the surface morphologies of the Mn oxides. Finally, these discoveries contribute to our understanding of how various Mn oxides form in nature and provide insights into the processes involved in manufacturing specific Mn oxide crystalline structures for engineering applications.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Redox cycling-based detection of phenazine metabolites secreted from Pseudomonas aeruginosa in nanopore electrode arrays

The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) produces several redox-active phenazine metabolites, including pyocyanin (PYO) and phenazine-1-carboxamide (PCN), which are electron carrier molecules that also aid in virulence. In particular, PYO is an exclusive metabolite produced by P. aeruginosa, which acts as a virulence factor in hospital-acquired infections and is therefore a good biomarker for identifying early stage colonization by this pathogen. Here, we describe the use of nanopore electrode arrays (NEAs) exhibiting metal–insulator–metal ring electrode architectures for enhanced detection of these phenazine metabolites. The size of the nanopores allows phenazine metabolites to freely diffuse into the interior and access the working electrodes, while the bacteria are excluded. Consequently, highly efficient redox cycling reactions in the NEAs can be accessed by free diffusion unhindered by the presence of bacteria. This strategy yields low limits of detection, i.e. 10.5 and 20.7 nM for PYO and PCN, respectively, values far below single molecule pore occupancy, e.g. at 10.5 nM < n pore > ~ 0.082 per nanopore – a limit which reflects the extraordinary signal amplification in the NEAs. Furthermore, experiments that compared results from minimal medium and rich medium show that P. aeruginosa produces the same types of phenazine metabolites even though growth rates and phenazine production patterns differ in these two media. Here, the NEA measurement strategy developed here should be useful as a diagnostic for pathogens generally and for understanding metabolism in clinically important microbial communities.

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

Nutrient Inputs Stimulate Mercury Methylation by Syntrophs in a Subarctic Peatland

Climate change dramatically impacts Arctic and subarctic regions, inducing shifts in wetland nutrient regimes as a consequence of thawing permafrost. Altered hydrological regimes may drive changes in the dynamics of microbial mercury (Hg) methylation and bioavailability. Important knowledge gaps remain on the contribution of specific microbial groups to methylmercury (MeHg) production in wetlands of various trophic status. Here, we measured aqueous chemistry, potential methylation rates (k meth ), volatile fatty acid (VFA) dynamics in peat-soil incubations, and genetic potential for Hg methylation across a groundwater-driven nutrient gradient in an interior Alaskan fen. We tested the hypotheses that (1) nutrient inputs will result in increased methylation potentials, and (2) syntrophic interactions contribute to methylation in subarctic wetlands. We observed that concentrations of nutrients, total Hg, and MeHg, abundance of hgcA genes, and rates of methylation in peat incubations (k meth ) were highest near the groundwater input and declined downgradient. hgcA sequences near the input were closely related to those from sulfate-reducing bacteria (SRB), methanogens, and syntrophs. Hg methylation in peat incubations collected near the input source (FPF2) were impacted by the addition of sulfate and some metabolic inhibitors while those down-gradient (FPF5) were not. Sulfate amendment to FPF2 incubations had higher k meth relative to unamended controls despite no effect on k meth from addition of the sulfate reduction inhibitor molybdate. The addition of the methanogenic inhibitor BES (25 mM) led to the accumulation of VFAs, but unlike molybdate, it did not affect Hg methylation rates. Rather, the concurrent additions of BES and molybdate significantly decreased k meth , suggesting a role for interactions between SRB and methanogens in Hg methylation. The reduction in k meth with combined addition of BES and molybdate, and accumulation of VFA in peat incubations containing BES, and a high abundance of syntroph-related hgcAsequences in peat metagenomes provide evidence for MeHg production by microorganisms growing in syntrophy. Collectively the results suggest that wetland nutrient regimes influence the activity of Hg methylating microorganisms and, consequently, Hg methylation rates. Our results provide key information about microbial Hg methylation and methylating communities under nutrient conditions that are expected to become more common as permafrost soils thaw.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Bacterial xylan utilization regulons: systems for coupling depolymerization of methylglucuronoxylans with assimilation and metabolism

Abstract Bioconversion of lignocellulosic resources offers an economically promising path to renewable energy. Technological challenges to achieving bioconversion include the development of cost-effective processes that render the cellulose and hemicellulose components of these resources to fermentable hexoses and pentoses. Natural bioprocessing of the hemicellulose fraction of lignocellulosic biomass requires depolymerization of methylglucuronoxylans. This requires secretion of endoxylanases that release xylooligosaccharides and aldouronates. Physiological, biochemical, and genetic studies with selected bacteria support a process in which a cell-anchored multimodular GH10 endoxylanase catalyzes release of the hydrolysis products, aldotetrauronate, xylotriose, and xylobiose, which are directly assimilated and metabolized. Gene clusters encoding intracellular enzymes, including α-glucuronidase, endoxylanase, β-xylosidase, ABC transporter proteins, and transcriptional regulators, are coordinately responsive to substrate induction or repression. The rapid rates of glucuronoxylan utilization and microbial growth, along with the absence of detectable products of depolymerization in the medium, indicate that assimilation and depolymerization are coupled processes. Genomic comparisons provide evidence that such systems occur in xylanolytic species in several genera, including Clostridium, Geobacillus, Paenibacillus, and Thermotoga. These systems offer promise, either in their native configurations or through gene transfer to other organisms, to develop biocatalysts for efficient production of fuels and chemicals from the hemicellulose fractions of lignocellulosic resources.

Biotechnology & Applied Microbiology↗
DOE OSTI2021

Integrating continuous hypermutation with high–throughput screening for optimization of cis,cis –muconic acid production in yeast

Directed evolution is a powerful method to optimize proteins and metabolic reactions towards user-defined goals. It usually involves subjecting genes or pathways to iterative rounds of mutagenesis, selection and amplification. While powerful, systematic searches through large sequence-spaces is a labour-intensive task, and can be further limited by a priori knowledge about the optimal initial search space, and/or limits in terms of screening throughput. Here, we demonstrate an integrated directed evolution workflow for metabolic pathway enzymes that continuously generate enzyme variants using the recently developed orthogonal replication system, OrthoRep and screens for optimal performance in high-throughput using a transcription factor-based biosensor. We demonstrate the strengths of this workflow by evolving a rate-limiting enzymatic reaction of the biosynthetic pathway for cis,cis-muconic acid (CCM), a precursor used for bioplastic and coatings, in Saccharomyces cerevisiae. After two weeks of simply iterating between passaging of cells to generate variant enzymes via OrthoRep and high-throughput sorting of best-performing variants using a transcription factor-based biosensor for CCM, we ultimately identified variant enzymes improving CCM titers > 13-fold compared with reference enzymes. Taken together, the combination of synthetic biology tools as adopted in this study is an efficient approach to debottleneck repetitive workflows associated with directed evolution of metabolic enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Inducible flippase-mediated metabolic engineering of Rhodosporidium toruloides for enhanced 3-hydroxypropionic acid production from corn stover hydrolysate

Rhodosporidium toruloides has gained increasing interests as a promising non-model host organism to produce a wide range of bioproducts from lignocellulosic biomass. Increasing the bioproduct titers, rates, and yields remains a challenge, largely due to a lack of robust and well-characterized genetic tools in this host. Here we developed an inducible flippase (FLP) and flippase recognition target (FRT) system that enables genetic manipulations without the need for additional selection markers. Synthetic inducible promoters were established, enabling regulation of FLP expression and efficient antibiotic marker removal. Leveraging this system, we engineered a strain to optimize 3-hydroxypropionic acid (3HP) production. Over four rounds of iterative genomic editing to resolve pathway bottlenecks, we achieved a 3HP titer of 69.4 g/L in fed-batch fermentation - the highest level reported in yeast from lignocellulosic hydrolysates. The engineered high 3HP producing strain offers a robust platform for sustainable bio-based chemical production from lignocellulosic feedstocks.

3-hydroxypropionic acid↗