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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 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Data for Rewiring Yeast Metabolism for Producing 2,3-Butanediol and Two Downstream Applications: Techno-Economic Analysis and Life Cycle Assessment of Methyl Ethyl Ketone (MEK) and Agricultural Biostimulant Production

Rising concerns for sustainability and global climate change have driven the development of sustainable production pathways for biofuels and chemicals from lignocellulosic biomass via integrated biological and chemical processes. We constructed an engineered Saccharomyces cerevisiae capable of producing 2,3-butanediol (2,3-BDO) from glucose without accumulating ethanol and glycerol, which hinder downstream processing of 2,3-BDO, through extensive metabolic reprogramming. Specifically, we introduced heterologous 2,3-BDO biosynthetic enzymes and deleted the major isozymes of ethanol and glycerol biosynthetic enzymes. In addition, we introduced an NAD+ regenerating Pyruvate-Malate (PM) cycle and enhanced the NAD+ regenerating capability of the PM cycle to resolve the redox imbalance from the deletion of ethanol and glycerol production pathways. The resulting engineered yeast produced 109.9 g/L of 2,3-BDO with a productivity of 1.0 g/L/h and a yield of 0.36 g/g glucose in a fed-batch fermentation. We also conducted techno-economic analysis (TEA) and life cycle assessment (LCA) of the production of methyl ethyl ketone (MEK) through catalytic dehydration of 2,3-BDO. A TEA based on the experimental results indicated that the minimum product selling price (MPSP) was estimated to be $1.90/kg. Regarding cradle-to-grave LCA, 100-year global warming potential (GWP100) and fossil energy consumption (FEC) were found to be 0.37 kg CO2 eq/kg and 3.1 MJ/kg, respectively. These results demonstrated the feasibility of cost-competitive and sustainable bio-based MEK production via yeast fermentation. In addition, we explored the possibility of using the fermentation broth containing 2,3-BDO as a biostimulant inducing drought tolerance in plants. As a result, the yeast 2,3-BDO fermentation broth can induce drought tolerance in Arabidopsis thaliana without a complicated purification process.

Economics↗
DOE OSTI2021

Integration of proteomics and metabolomics into the Design, Build, Test, Learn cycle to improve 3-hydroxypropionic acid production in Aspergillus pseudoterreus

Biological engineering of microorganisms to produce value-added chemicals is a promising route to sustainable manufacturing. However, overproduction of metabolic intermediates at high titer, rate, and yield from inexpensive substrates is challenging in non-model systems where limited information is available regarding metabolic flux and its control in production conditions. Integrated multi-omic analyses of engineered strains offers an in-depth look at metabolites and proteins directly involved in growth and production of target and non-target bioproducts. Here we applied multi-omic analyses to overproduction of the polymer precursor 3-hydroxypropionic acid (3HP) in the filamentous fungus Aspergillus pseudoterreus. A synthetic pathway consisting of aspartate decarboxylase, beta-alanine pyruvate transaminase, and 3HP dehydrogenase was designed and built for A. pseudoterreus. Strains with single- and multi-copy integration events were isolated and multi-omics analysis consisting of intracellular and extracellular metabolomics and targeted and global proteomics was used to interrogate the strains in shake-flask and bioreactor conditions. Production of a variety of co-products (organic acids and glycerol) and oxidative degradation of 3HP were identified as metabolic pathways competing with 3HP production. Intracellular accumulation of nitrogen as 2,4-diaminobutanoate was identified as an off-target nitrogen sink that may also limit flux through the engineered 3HP pathway. Elimination of the high-expression oxidative 3HP degradation pathway by deletion of a putative malonate semialdehyde dehydrogenase improved the yield of 3HP by 3.4× after 10 days in shake-flask culture. This is the first report of 3HP production in a filamentous fungus amenable to industrial scale biomanufacturing of organic acids at high titer and low pH.

Pomraning, Kyle R.↗
DOE OSTI2021

A Genome-Scale Metabolic Model of Anabaena 33047 to Guide Genetic Modifications to Overproduce Nylon Monomers

Nitrogen fixing-cyanobacteria can significantly improve the economic feasibility of cyanobacterial production processes by eliminating the requirement for reduced nitrogen. Anabaena sp. ATCC 33047 is a marine, heterocyst forming, nitrogen fixing cyanobacteria with a very short doubling time of 3.8 h. We developed a comprehensive genome-scale metabolic (GSM) model, iAnC892, for this organism using annotations and content obtained from multiple databases. iAnC892 describes both the vegetative and heterocyst cell types found in the filaments of Anabaena sp. ATCC 33047. iAnC892 includes 953 unique reactions and accounts for the annotation of 892 genes. Comparison of iAnC892 reaction content with the GSM of Anabaena sp. PCC 7120 revealed that there are 109 reactions including uptake hydrogenase, pyruvate decarboxylase, and pyruvate-formate lyase unique to iAnC892. iAnC892 enabled the analysis of energy production pathways in the heterocyst by allowing the cell specific deactivation of light dependent electron transport chain and glucose-6-phosphate metabolizing pathways. The analysis revealed the importance of light dependent electron transport in generating ATP and NADPH at the required ratio for optimal N2 fixation. When used alongside the strain design algorithm, OptForce, iAnC892 recapitulated several of the experimentally successful genetic intervention strategies that over produced valerolactam and caprolactam precursors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Microorganisms that co-consume glucose with non-glucose carbohydrates and methods of use

Microorganisms that co-consume glucose with non-glucose carbohydrates, such as xylose, and methods of using same. The microorganisms comprise modifications that reduce or ablate the activity of a phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) protein or modifications that reduce or ablate the activity of a phosphoglucose isomerase and a GntR. The PTS protein may be selected from an enzyme I (EI), an HPr, an FPr, and an enzyme IIGlc (EIIGlc). Additional modifications include reduction or ablation of the activity of a pyruvate formate lyase, a lactate dehydrogenase, and a fumarate reductase and inclusion of recombinant pyruvate decarboxylase and alcohol dehydrogenase genes. The microorganisms are particularly suited to co-consuming glucose and xylose in media containing these substrates and producing ethanol therefrom.

Reed, Jennifer L.↗
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 OSTI2023

Uncovering the dominant role of root metabolism in shaping rhizosphere metabolome under drought in tropical rainforest plants

Plant-soil-microbe interactions are crucial for driving rhizosphere processes that contribute to metabolite turnover and nutrient cycling. With the increasing frequency and severity of water scarcity due to climate warming, understanding how plant-mediated processes, such as root exudation, influence soil organic matter turnover in the rhizosphere is essential. In this study, we used 16S rRNA gene amplicon sequencing, rhizosphere metabolomics, and position-specific 13 C-pyruvate labeling to examine the effects of three different plant species (Piper auritum, Hibiscus rosa sinensis, and Clitoria fairchildiana) and their associated microbial communities on soil organic carbon turnover in the rhizosphere. Here, our findings indicate that in these tropical plants, the rhizosphere metabolome is primarily shaped by the response of roots to drought rather than direct shifts in the rhizosphere bacterial community composition. Specifically, the reduced exudation of plant roots had a notable effect on the metabolome of the rhizosphere of P. auritum, with less reliance on neighboring microbes. Contrary to P. auritum, H. rosa sinensis and C. fairchildiana experienced changes in their exudate composition during drought, causing alterations to the bacterial communities in the rhizosphere. This, in turn, had a collective impact on the rhizosphere's metabolome. Furthermore, the exclusion of phylogenetically distant microbes from the rhizosphere led to shifts in its metabolome. Additionally, C. fairchildiana appeared to be associated with only a subset of symbiotic bacteria under drought conditions. These results indicate that plant species-specific microbial interactions systematically change with the root metabolome. As roots respond to drought, their associated microbial communities adapt, potentially reinforcing the drought tolerance strategies of plant roots. These findings have significant implications for maintaining plant health and preference during drought stress and improving plant performance under climate change.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Transcriptomic and metabolomic changes in postharvest sugarbeet roots reveal widespread metabolic changes in storage and identify genes potentially responsible for respiratory sucrose loss

Endogenous metabolism is primarily responsible for losses in sucrose content and processing quality in postharvest sugarbeet roots. The genes responsible for this metabolism and the transcriptional changes that regulate it, however, are largely unknown. To identify genes and metabolic pathways that participate in postharvest sugarbeet root metabolism and the transcriptional changes that contribute to their regulation, transcriptomic and metabolomic profiles were generated for sugarbeet roots at harvest and after 12, 40 and 120 d storage at 5 and 12°C and gene expression and metabolite concentration changes related to storage duration or temperature were identified. During storage, 8656 genes, or 34% of all expressed genes, and 225 metabolites, equivalent to 59% of detected metabolites, were altered in expression or concentration, indicating extensive transcriptional and metabolic changes in stored roots. These genes and metabolites contributed to a wide range of cellular and molecular functions, with carbohydrate metabolism being the function to which the greatest number of genes and metabolites classified. Because respiration has a central role in postharvest metabolism and is largely responsible for sucrose loss in sugarbeet roots, genes and metabolites involved in and correlated to respiration were identified. Seventy-five genes participating in respiration were differentially expressed during storage, including two bidirectional sugar transporter SWEET17 genes that highly correlated with respiration rate. Weighted gene co-expression network analysis identified 1896 additional genes that positively correlated with respiration rate and predicted a pyruvate kinase gene to be a central regulator or biomarker for respiration rate. Overall, these results reveal the extensive and diverse physiological and metabolic changes that occur in stored sugarbeet roots and identify genes with potential roles as regulators or biomarkers for respiratory sucrose loss.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Organic Acid Aerosol Measurements from the Mount Airy Site for CoURAGE

This study investigates the prevalence and distribution of organic acid aerosols in a rural environment using filter-based measurements collected in Mount Airy, Maryland, during the CoURAGE campaign from March 19th through June 12th 2025. PM2.5 filters quantify a range of organic acids commonly associated with secondary organic aerosol formation and atmospheric oxidation processes. Each filter was collected using a 15 LPM sampler and was extracted in ultrapure Millipore water (>18 MΩ), allowing water-soluble organic acids to be extracted into solution for analysis. The extracts were then examined using a Waters Acquity I-Class PLUS liquid chromatography system coupled to a Bruker Maxis-II ultra-high-resolution Q-TOF mass spectrometer with electrospray ionization, providing high-sensitivity detection and separation of target compounds. Concentrations of several key organic acids were quantified, including acetic, propionic, pyruvic, butyric, oxalic, isovaleric, valeric, malonic, maleic, succinic, glutaric, malic, adipic, and citric acids. These findings contribute to ongoing efforts to understand regional aerosol composition and their impacts on aerosol-cloud interactions.

Acetic acid↗
DOE OSTI2024

Toward Ultra‐High‐Quality‐Factor Wireless Masing Magnetic Resonance Sensing

It has recently been shown that a bolus of hyperpolarized nuclear spins can yield stimulated emission signals similar in nature to maser signals, potentially enabling new ways of sensing hyperpolarized contrast media, including most notably [1- 13 C]pyruvate that is under evaluation in over 50 clinical trials for metabolic imaging of cancer. The stimulated NMR signal emissions lasting for minutes do not require radio-frequency excitation, offering unprecedented advantages compared to conventional MR sensing. However, creating nuclear spin maser emission is challenging in practice due to stringent fundamental requirements, making practical in vivo applications hardly possible using conventional passive MR detectors. Here, in this study, we demonstrate the utility of a wireless NMR maser detector, the quality factor of which was enhanced 22-fold (to 1,670) via parametric pumping. This active-feedback technique breaks the intrinsic fundamental limit of NMR detector circuit quality factor. We show the use of parametric pumping to reduce the threshold requirement for inducing nuclear spin masing at 300 MHz resonance frequency in a preclinical MRI scanner. Indeed, stimulated emission from hyperpolarized protons was obtained under highly unfavorable conditions of low magnetic field homogeneity (T 2 * of 3 ms). Greater gains of the quality factor of the MR detector (up to 1 million) were also demonstrated.

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

L–malic acid production from xylose by engineered Saccharomyces cerevisiae

L-malic acid is widely used in the food, chemical, and pharmaceutical industries. Here, we report on production of malic acid from xylose, the second most abundant sugar in lignocellulosic hydrolysates, by engineered Saccharomyces cerevisiae. To enable malic acid production in a xylose-assimilating S. cerevisiae, we overexpressed PYC1 and PYC2, coding for pyruvate carboxylases, a truncated MDH3 coding for malate dehydrogenase, and SpMAE1, coding for a Schizosaccharomyces pombe malate transporter. Additionally, both the ethanol and glycerol-producing pathways were blocked to enhance malic acid production. The resulting strain produced malic acid from both glucose and xylose, but it produced much higher titers of malic acid from xylose than glucose. Interestingly, the engineered strain had higher malic acid yield from lower concentrations (10 g L –1 ) of xylose, with no ethanol production, than from higher xylose concentrations (20 and 40 g L –1 ). As such, a fed-batch culture maintaining xylose concentrations at low levels was conducted and 61.2 g L –1 of malic acid was produced, with a productivity of 0.32 g L –1 h. Furthermore, these results represent successful engineering of S. cerevisiae for the production of malic acid from xylose, confirming that that xylose offers the efficient production of various biofuels and chemicals by engineered S. cerevisiae.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Cloning and characterization of a panel of mitochondrial targeting sequences for compartmentalization engineering in Saccharomyces cerevisiae

Mitochondrion is generally considered as the most promising subcellular organelle for compartmentalization engineering. Much progress has been made in reconstituting whole metabolic pathways in the mitochondria of yeast to harness the precursor pools (i.e., pyruvate and acetyl-CoA), bypass competing pathways, and minimize transportation limitations. However, only a few mitochondrial targeting sequences (MTSs) have been characterized (i.e., MTS of COX4), limiting the application of compartmentalization engineering for multigene biosynthetic pathways in the mitochondria of yeast. In the present study, based on the mitochondrial proteome, a total of 20 MTSs were cloned and the efficiency of these MTSs in targeting heterologous proteins, including the Escherichia coli FabI and enhanced green fluorescence protein (EGFP) into the mitochondria was evaluated by growth complementation and confocal microscopy. After systematic characterization, six of the well!performed MTSs were chosen for the colocalization of complete biosynthetic pathways into the mitochondria. As proof of concept, the full alpha-santalene biosynthetic pathway consisting of 10 expression cassettes capable of converting acety coA to alpha-santalene was compartmentalized into the mitochondria, leading to a 3.7 fold improvement in the production of alpha-santalene. Furthermore, the newly characterized MTSs should contribute to the expanded metabolic engineering and synthetic biology toolbox for yeast mitochondrial compartmentalization engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Thirdhand cigarette smoke leads to age-dependent and persistent alterations in the cecal microbiome of mice

The gut microbiome composition is influenced by many factors including environmental exposures. Here, we investigated the effect of thirdhand cigarette smoke (THS) and exposure age on gut microbiome diversity. C57BL/6 mice were exposed to THS at human exposure relevant levels for three weeks during three different life stages: postnatal (0–3 weeks of age), pubescent (4–7 weeks of age), and adult (9–12 weeks of age), respectively. Cecal microbiome profiles were assessed at 17 weeks of age by 16S rRNA gene sequencing. We found that age at THS exposure strongly influenced the cecal microbiome composition. Although postnatal THS exposure significantly influenced the microbial composition, pubescent and adulthood exposures only had minor effects. The microbiome of postnatally THS-exposed mice significantly increased several degradation pathways that regulate glycolysis and pyruvate decarboxylation, and significantly decreased coenzyme A biosynthesis and pyrimidine deoxyribonucleoside salvage. Our results indicate that mouse postnatal development is particularly susceptible to persistent THS exposure effects on the gut microbiome.

16S rRNA gene sequencing↗
DOE OSTI2021

High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics

Membrane proteins play essential roles in cellular function and metabolism. Nonetheless, biophysical and structural studies of membrane proteins are impeded by the difficulty of their expression in and purification from heterologous cell-based systems. As an alternative to these cell-based systems, cell-free protein synthesis has proven to be an exquisite method for screening membrane protein targets in a variety of lipidic mimetics. Here we report a high-throughput screening workflow and applied it to screen 61eukaryotic membrane protein targets. For each target, we tested its expression in lipidic mimetics: two detergents, two liposomes, and two nanodiscs. We show that 35 membrane proteins (57%) can be expressed in a soluble fraction in at least one of the mimetics with the two detergents performing significantly better than nanodiscs and liposomes, in that order. Using the established cell-free workflow, we studied the production and biophysical assays for mitochondrial pyruvate carrier (MPC) complexes. Our studies show that the complexes produced in cell-free are functional competent in complex formation and substrate binding. Our results highlight the utility of using cell-free systems for screening and production of eukaryotic membrane proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Identification of a key water molecule involved in the macrophage migration inhibitory factor‐catalyzed tautomerization of para ‐hydroxyphenylpyruvate using neutron crystallography

Abstract Neutron crystallography was used to determine a 2.5‐Å resolution all‐atom structure of macrophage migration inhibitory factor (MIF) interacting with 3‐(4‐hydroxyphenyl)‐pyruvate (HPP). MIF is a pro‐inflammatory, pro‐tumorigenic protein that may be an attractive therapeutic target. MIF catalyzes the interconversion of the keto and enol forms of HPP by a tautomerase reaction. Although HPP is evidently not a physiological substrate of MIF, many compounds that inhibit this activity in enzymatic assays have been found also to inhibit physiological activities of MIF. Therefore, the MIF‐catalyzed HPP tautomerization reaction is used in initial screening of compounds in the search for inhibitors of MIF physiological activity. The neutron diffraction‐derived crystal structure reveals the position of a water molecule involved in the tautomerization reaction, and also confirms the charged state of lysine‐32 in the active site. The structure confirms the previously proposed catalytic mechanism of MIF, with the N‐terminal Pro‐1 abstracting a proton to generate an HPP enolate intermediate which is subsequently protonated. The structure reported herein reveals that this proton is supplied by a neighboring water molecule. Along with the neutron structure, a room‐temperature synchrotron x‐ray crystal structure reveals a covalent adduct between HPP and MIF. While this adduct is a result of radiation‐induced chemistry, its formation confirms the catalytic role of the active site residue because a covalent complex could only form if the reactive carbon of the substrate is correctly positioned by the enzyme.

Schröder, Gabriela C. [Department of Molecular and↗
DOE OSTI2021

Investigating the role of the transcriptional regulator Ure2 on the metabolism of Saccharomyces cerevisiae: a multi-omics approach

Ure2 regulates nitrogen catabolite repression in Saccharomyces cerevisiae. Deletion of URE2 induces a physiological state mimicking the nitrogen starvation and autophagic responses. Previous work has shown that deletion of URE2 increases the fermentation rate of some wine-producing strains of S. cerevisiae. In this work, we investigated the effect of URE2 deletion (ΔURE2) on the metabolism of S. cerevisiae. During growth on glucose, the ΔURE2 mutant grew less well than the wild type; however, it produced ethanol at higher rates. To better under the behavior of this mutant, we performed transcriptomics and metabolomics. Analysis of the RNA sequencing results and metabolite levels indicate that the mutant strain exhibited characteristics of both nitrogen starvation and autophagy. In addition, many pyruvate decarboxylase and alcohol dehydrogenase isoforms were expressed at higher rates than the wild type. The mutant also accumulated less trehalose and glycogen, and produced more lipids. These results suggest that URE2 may be a promising target for metabolic engineering in S. cerevisiae and potentially other strains of yeast as well for the production of lipid-based fuels and chemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Time-resolved targeted metabolomics shows an abrupt switch from Calvin-Benson-Bassham cycle to tricarboxylic acid cycle when the light is turned off

Abstract In leaves, major CO₂ fluxes alternate between fixation by the Calvin-Benson-Bassham (CBB) cycle during light and release by the tricarboxylic acid (TCA) cycle in darkness. The speed at which leaf metabolism transitions between these pathways likely influences plant tolerance to fluctuating light conditions. To investigate these rapid metabolic shifts, we exposed leaves to ¹³CO₂ for 20 min to establish a quasi-steady state before abruptly turning off the light while maintaining ¹³CO₂ feeding. Within 10 s of dark transition, 3-phosphoglycerate levels rose significantly while most other CBB cycle intermediates decreased by more than 90%. Simultaneously, carbon accumulated in alanine, likely via pyruvate. Over the subsequent 10 min, six- and five-carbon TCA cycle intermediates steadily increased. In contrast, four-carbon TCA intermediates peaked at one minute, declined at three minutes, and rose again at 10 min, a pattern mirrored by most measured amino acids. These results reveal an exceptionally rapid metabolic reconfiguration from CO₂ fixation by the CBB cycle in light to TCA cycle activation for energy production in darkness, accompanied by substantial changes in amino acid metabolism.

Plant Sciences↗
DOE OSTI2022

A mutagenic analysis of NahE, a hydratase-aldolase in the naphthalene degradative pathway

NahE is a hydratase-aldolase that converts o-substituted trans-benzylidenepyruvates (H, OH, or CO 2 - ) to benzaldehyde, salicylaldehyde, or 2-carboxybenzaldehyde, respectively, and pyruvate. The enzyme is in a bacterial degradative pathway for naphthalene, which is a toxic and persistent environmental contaminant. Sequence, crystallographic, and mutagenic analysis identified the enzyme as a member of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. As such, it has a conserved lysine (Lys183) and tyrosine (Tyr155), for Schiff base formation, as well as a GXXGE motif for binding of the pyruvoyl carboxylate group. A crystal structure of the selenomethionine derivative of NahE shows these active site elements along with nearby residues that might be involved in the mechanism and/or specificity. Mutations of five active site amino acids (Thr65, Trp128, Tyr155, Asn157, and Asn281) were constructed and kinetic parameters measured in order to assess the effect(s) on catalysis. Here, the results show that the two Trp128 mutants (Phe and Tyr) have the least effect on catalysis, whereas amino acids with bulky side chains at Thr65 (Val) and Asn281 (Leu) have the greatest effect. Changing Tyr155 to Phe and Asn157 to Ala also hinders catalysis, and the effects fall in between these extremes. These observations are put into a structural context using a crystal structure of the Schiff base of the reaction intermediate. Trapping experiments with substrate, Na(CN)BH 3 , and wild type enzyme and selected mutants mostly paralleled the kinetic analysis, and identified two salicylaldehyde-modified lysines: the active site lysine (Lys183) and one outside the active site (Lys279). The latter could be responsible for the observed inhibition of NahE by salicylaldehyde. Together, the results provide new insights into the NahE-catalyzed reaction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Rewiring yeast metabolism for producing 2,3-butanediol and two downstream applications: Techno-economic analysis and life cycle assessment of methyl ethyl ketone (MEK) and agricultural biostimulant production

Rising concerns for sustainability and global climate change have driven the development of sustainable pro- duction pathways for biofuels and chemicals from lignocellulosic biomass via integrated biological and chemical processes. We constructed an engineered Saccharomyces cerevisiae capable of producing 2,3-butanediol (2,3-BDO) from glucose without accumulating ethanol and glycerol, which hinder downstream processing of 2,3-BDO, through extensive metabolic reprogramming. Specifically, we introduced heterologous 2,3-BDO biosynthetic enzymes and deleted the major isozymes of ethanol and glycerol biosynthetic enzymes. In addition, we introduced an NAD + regenerating Pyruvate-Malate (PM) cycle and enhanced the NAD + regenerating capability of the PM cycle to resolve the redox imbalance from the deletion of ethanol and glycerol production pathways. The resulting engineered yeast produced 109.9 g/L of 2,3-BDO with a productivity of 1.0 g/L/h and a yield of 0.36 g/ g glucose in a fed-batch fermentation. We also conducted techno-economic analysis (TEA) and life cycle assessment (LCA) of the production of methyl ethyl ketone (MEK) through catalytic dehydration of 2,3-BDO. A TEA based on the experimental results indicated that the minimum product selling price (MPSP) was estimated to be $1.90/kg. Regarding cradle-to-grave LCA, 100-year global warming potential (GWP100) and fossil energy consumption (FEC) were found to be 0.37 kg CO 2 eq/kg and 3.1 MJ/kg, respectively. These results demonstrated the feasibility of cost-competitive and sustainable bio-based MEK production via yeast fermentation. In addition, we explored the possibility of using the fermentation broth containing 2,3-BDO as a biostimulant inducing drought tolerance in plants. As a result, the yeast 2,3-BDO fermentation broth can induce drought tolerance in Arabidopsis thaliana without a complicated purification process.

2,3-butanediol (2,3-BDO)↗