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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Engineering and evolution of Yarrowia lipolytica for producing lipids from lignocellulosic hydrolysates

Yarrowia lipolytica, an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

raogroupuiuc/yl_variantcalling

NGS analysis for mutation analysis in Yarrowia lipolytica evolved strains. Yarrowia lipolytica, an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates.

Deewan, Anshu↗
DOE OSTI2025

Protein carbamylation and proteomics: from artifacts to elucidation of biological functions

Lysine carbamylation is a non-enzymatic protein post-translational modification (PTM) that plays important roles in regulating enzymatic activity and the pathogenesis of diseases such as atherosclerosis, rheumatoid arthritis, and uremia. The progress of understanding the roles of carbamylation in biological systems has been delayed due to lack of systematic assays to study its functions. To aggravate this scenario, carbamylation is a major artifact in proteomics analysis given that urea, which is used during sample preparation, induces carbamylation. In addition, anti-acetyllysine antibodies co-purify carbamylated and acetylated peptides. In a recent paper, we leveraged co-purification with anti-acetyllysine antibodies to develop a method for analyzing carbamylated proteomes. In this perspective article, we discuss how this method may be applied to characterize the physiological functions of carbamylation in humans and other biological models, as well as the utility of establishing novel disease biomarkers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

EGLP-1 lowers body weight better than exendin-4 by reducing food intake and increasing basal energy expenditure in diet-induced obese mice

It is well known that GLP-1 activates GLP-1R to reduce body weight by inhibiting eating. GLP-1 is cleaved by the neutral endopeptidase (NEP) 24.11 into a pentapeptide GLP-1 (32–36) amide, which increases basal energy expenditure and inhibits weight gain in obese mice. It is well known that GLP-1 analogs can reduce weight by suppressing eating. However, there are few reports of reducing weight through the dual effects of inhibiting eating and increasing basic energy. Here, we report the peptide EGLP-1, a GLP-1 analogue, which can reduce food intake and increase basal energy expenditure. In C2C12 myotubes, EGLP-1 can increase both phosphorylation of acetyl CoA carboxylase (ACC) and the ratio between phosphorylation of ACC and the total expression of ACC (pACC/ACC). In diet-induced obese mice, EGLP-1 is more effective than exendin-4 in reducing body weight, reducing fat mass and improving hepatic steatosis. At the same time, EGLP-1 can improve hyperglycemia, reduce food intake, and improve insulin resistance, just like exendin-4. In addition, EGLP-1, not exendin-4, can improve physiological parameters associated with lipid metabolism and increase oxygen consumption by increasing uncoupling proteins 3 (UCP3) expression and pACC/ACC ratio in skeletal muscle. Taken together, this data showed that EGLP-1 is able to reduce body weight by reducing food intake and increasing basal energy expenditure, suggesting it may be more effective in treating diabetic and non-diabetic overweight or obese people than pure GLP-1R agonist exendin-4.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1995

Two cis elements collaborate to spatially repress transcription from a sea urchin promoter

The expression pattern of many territory-specific genes in metazoan embryos is maintained by an active process of negative spatial regulation. However, the mechanism of this strategy of gene regulation is not well understood in any system. Here we show that reporter constructs containing regulatory sequence for the SM30-alpha gene of Stronglyocentrotus purpuratus are expressed in a pattern congruent with that of the endogenous SM30 gene(s), largely as a result of active transcriptional repression in cell lineages in which the gene is not normally expressed. Chloramphenicol acetyl transferase assays of deletion constructs from the 2600-bp upstream region showed that repressive elements were present in the region from -1628 to -300. In situ hybridization analysis showed that the spatial fidelity of expression was severely compromised when the region from -1628 to -300 was deleted. Two highly repetitive sequence motifs, (G/A/C)CCCCT and (T/C)(T/A/C)CTTTT(T/A/C), are present in the -1628 to -300 region. Representatives of these elements were analyzed by gel mobility shift experiments and were found to interact specifically with protein in crude nuclear extracts. When oligonucleotides containing either sequence element were co-injected with a correctly regulated reporter as potential competitors, the reporter was expressed in inappropriate cells. When composite oligonucleotides, containing both sequence elements, were fused to a misregulated reporter, the expression of the reporter in inappropriate cells was suppressed. Comparison of composite oligonucleotides with oligonucleotides containing single constituent elements show that both sequence elements are required for effective spatial regulation. Thus, both individual elements are required, but only a composite element containing both elements is sufficient to function as a tissue-specific repressive element.

Non-NASA Center↗
DOE OSTI2021

An Improved Top-Down Mass Spectrometry Characterization of Chlamydomonas reinhardtii Histones and Their Post-translational Modifications

We present an updated analysis of the linker and core histone proteins and their proteoforms in the green microalga Chlamydomonas reinhardtii by top-down mass spectrometry (TDMS). The combination of high-resolution liquid chromatographic separation, robust fragmentation high, mass spectral resolution, the application of a custom search algorithm, and extensive manual analysis enabled the characterization of 86 proteoforms across all four core histones H2A, H2B, H3 and H4, and the linker histone H1. All canonical H2A paralogs, which vary in their C-termini, were identified, along with an unreported non-canonical variant that had both high levels of acetylation and C-terminal truncations. Similarly, a majority of the canonical H2B paralogs were identified, along with a smaller non-canonical variant, H2B.v, that was highly acetylated. Histone H4 exhibited a novel acetylation profile that differs significantly from that found in other organisms. A majority of H3 was monomethylated at K4, while a small fraction of H3 was trimethylated at K4 and also highly acetylated.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2008

Nanoflow Separation of Amino Acids for the Analysis of Cosmic Dust

The delivery of amino acids to the early Earth by interplanetary dust particles, comets, and carbonaceous meteorites could have been a significant source of the early Earth's prebiotic organic inventory. Amino acids are central to modern terrestrial biochemistry as major components of proteins and enzymes and were probably vital in the origin of life. A variety of amino acids have been detected in the CM carbonaceous meteorite Murchison, many of which are exceptionally rare in the terrestrial biosphere including a-aminoisobutyric acid (AIB) and isovaline. AIB has also been detected in a small percentage of Antarctic micrometeorite grains believed to be related to the CM meteorites We report on progress in optimizing a nanoflow liquid chromatography separation system with dual detection via laser-induced-fluorescence time of flight mass spectrometry (nLC-LIF/ToF-MS) for the analysis of o-phthaldialdehydelN-acetyl-L-cysteine (OPA/NAC) labeled amino acids in cosmic dust grains. The very low flow rates (<3 micro-L/min) of nLC over analytical LC (>0.1 ml/min) combined with <2 micron column bead sizes has the potential to produce efficient analyte ionizations andchromatograms with very sharp peaks; both increase sensitivity. The combination of the selectivity (only primary amines are derivatized), sensitivity (>4 orders of magnitude lower than traditional GC-MS techniques), and specificity (compounds identities are determined by both retention time and exact mass) makes this a compelling technique. However, the development of an analytical method to achieve separation of compounds as structurally similar as amino acid monomers and produce the sharp peaks required for maximum sensitivity is challenging.

Martin, M. P.↗
NASA NTRS2003

Structural Basis for "Flip-Flop" Action of Human Pyruvate Dehydrogenase

The derivative of vitamin B1, thiamin pyrophosphate is a cofactor of pyruvate dehydrogenase, a component enzyme of the mitochondrial pyruvate dehydrogenase multienzyme complex that plays a major role in directing energy metabolism in the cell. This cofactor is used to cleave the C(sup alpha)-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. In alpha(sub 2)beta(sub 2)-tetrameric human pyruvate dehydrogenase, there are two cofactor binding sites, each of them being a center of independently conducted, although highly coordinated enzymatic reactions. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites can now be understood based on the recently determined crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95A resolution. The structure of pyruvate dehydrogenase was determined using a combination of MAD phasing and molecular replacement followed by rounds of torsion-angles molecular-dynamics simulated-annealing refinement. The final pyruvate dehydrogenase structure included coordinates for all protein amino acids two cofactor molecules, two magnesium and two potassium ions, and 742 water molecules. The structure was refined to R = 0.202 and R(sub free) = 0.244. Our structural analysis of the enzyme folding and domain assembly identified a simple mechanism of this protein motion required for the conduct of catalytic action.

Ciszak, Ewa↗
NASA NTRS1993

Multipole correction of atomic monopole models of molecular charge distribution. I. Peptides

The defects in atomic monopole models of molecular charge distribution have been analyzed for several model-blocked peptides and compared with accurate quantum chemical values. The results indicate that the angular characteristics of the molecular electrostatic potential around functional groups capable of forming hydrogen bonds can be considerably distorted within various models relying upon isotropic atomic charges only. It is shown that these defects can be corrected by augmenting the atomic point charge models by cumulative atomic multipole moments (CAMMs). Alternatively, sets of off-center atomic point charges could be automatically derived from respective multipoles, providing approximately equivalent corrections. For the first time, correlated atomic multipoles have been calculated for N-acetyl, N'-methylamide-blocked derivatives of glycine, alanine, cysteine, threonine, leucine, lysine, and serine using the MP2 method. The role of the correlation effects in the peptide molecular charge distribution are discussed.

NASA Discipline Exobiology↗
DOE OSTI2025

MarK, a Novosphingobium aromaticivorans kinase required for catabolism of multiple aromatic monomers

The aromatic compounds used in a variety of industrial products are currently obtained from nonrenewable petroleum sources. Alternatively, the plant polymer lignin is an abundant renewable source of aromatics, and its depolymerization generates a variety of products that can include acetovanillone, a vanillin derivative containing an acetyl side chain. The Alphaproteobacterium Novosphingobium aromaticivorans DSM12444 can metabolize several chemically modified aromatics in deconstructed lignin, but not acetovanillone. In this work, adaptive laboratory evolution identified a single amino acid change in the previously uncharacterized gene product Saro_1862 that is necessary and sufficient for N. aromaticivorans growth with acetovanillone as a sole growth substrate, as well as other aromatic monomers not metabolized by wild-type cells. We show that a glutamate (E) to lysine (K) substitution at amino acid residue 16 of Saro_1862 results in a ~1600-fold increase in the rate of ATP-dependent acetovanillone phosphorylation. We also find that recombinant Saro_1862 E16K phosphorylates several other aromatic compounds in vitro , defining the first reported catalytic activity for the widespread UPF0261 protein domain contained in Saro_1862. Thus, we propose naming Saro_1862 MarK, for multiple aromatic kinase. A 1.57 Å crystal structure of MarK E16K predicts that the E16K substitution lies in a potential ATP binding site, suggesting how this amino acid change increased catalytic activity. A search for homologs of MarK and other proteins required for acetovanillone degradation predicts that this pathway for aromatic metabolism exists throughout the bacterial phylogeny.

Novosphingobium↗
DOE OSTI2025

Expression of a Bacterial Trehalose 6-Phosphate Synthase Gene otsA in Camelina sativa Seeds Promotes the Channelling of Carbon Towards Oil Accumulation

Improving seed oil yield is essential for developing Camelina sativa as a sustainable biofuel crop. Fatty acid synthesis depends on the production of acetyl-CoA from photosynthetically derived sugars. Trehalose 6-phosphate (T6P), a proxy for sucrose availability, can link sugar status to plant growth and development. Synthesised by trehalose 6-phosphate synthase (TPS) from UDP-glucose and glucose-6-phosphate, T6P plays a regulatory role in metabolism. Our previous studies on Arabidopsis transgenic lines constitutively expressing the E. coli otsA (encoding TPS) showed increased T6P levels and seed triacylglycerol, along with stunted growth. In the present study we express otsA in camelina under the control of a seed-specific Phaseolin promoter. Seeds of the resulting transgenic lines accumulated high levels of T6P, and a 15%–20% increase in total fatty acids and triacylglycerol compared to wild-type. Molecular analysis showed the transgenic seeds had reduced SnRK1 activity, elevated WRI1 protein levels, and increased the levels of WRI1 and its target genes, along with enhanced rates of fatty acid synthesis that increased seed weights relative to wild type. Notably, the increase in oil did not affect seed protein levels but did reduce the soluble metabolite fraction. Crucially, seed-specific expression of otsA mitigated the growth defects associated with constitutive otsA expression, and the transgenic lines showed normal seed development and germination. These findings demonstrate that targeted T6P modulation via seed-specific otsA expression is an effective metabolic engineering strategy to boost oil production in camelina and potentially in other oilseed crops and bioenergy crops such as energycane, sorghum and miscanthus.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2000

Acetylated Lysozyme as Impurity in Lysozyme Crystals: Constant Distribution Coefficient

Hen egg white lysozyme (HEWL) was acetylated to modify molecular charge keeping the molecular size and weight nearly constant. Two derivatives, A and B, more and less acetylated, respectively, were obtained, separated, purified and added to the solution from which crystals of tetragonal HEWL crystals were grown. Amounts of the A or B impurities added were 0.76, 0.38 and 0.1 milligram per millimeter while HEWL concentration were 20, 30 and 40 milligram per milliliter. The crystals grown in 18 experiments for each impurity were dissolved and quantities of A or B additives in these crystals were analyzed by cation exchange high performance liquid chromatography. All the data for each set of 18 samples with the different impurity and regular HEWL concentrations is well described by one distribution coefficient K = 2.15 plus or minus 0.13 for A and K = 3.42 plus or minus 0.25 for B. The observed independence of the distribution coefficient on both the impurity concentration and supersaturation is explained by the dilution model described in this paper. It shows that impurity adsorption and incorporation rate is proportional to the impurity concentration and that the growth rate is proportional to the crystallizing protein in solution. With the kinetic coefficient for crystallization, beta = 5.10(exp -7) centimeters per second, the frequency at which an impurity molecule near the growing interface irreversibly joins a molecular site on the crystal was found to be 3 1 per second, much higher than the average frequency for crystal molecules. For best quality protein crystals it is better to have low microheterogeneous protein impurity concentration and high supers aturation.

Thomas, B. R.↗
NASA NTRS1994

Catalytic roles of the AMP at the 3' end of tRNAs

Recent reports suggest that the ribosome retains considerable peptidyl transferase activity even when much of the protein of the ribosome is removed and further suggests that rRNA may be the peptidyl transferase. The work here suggests that the AMP residue at the 3' terminus of each tRNA has some catalytic activity both in the esterification reaction and in forming a pseudopeptide, AcGly, and further suggests that whatever peptidyl transferase is, it finds a cooperative substrate in the aminoacyl-AMP at the 3' terminus of tRNA.

Non-NASA Center↗
DOE OSTI2021

Major involvement of two laccase genes in conidial pigment biosynthesis in Aspergillus oryzae

Wild-type strains of Aspergillus oryzae develop yellow, yellow-green, green, or brown conidia. Previous reports suggested that the conidiation initiates with the biosynthesis of a yellow pigment YWA1 from acetyl-CoA by a polyketide synthase encoded by wA (AO090102000545). This is followed by the conversion to other pigment by a laccase encoded by yA (AO090011000755). Based on orthologous pathways in other aspergilli, it is reasonable to hypothesize that in addition to yA, AO090102000546 encoding laccase and AO090005000332 encoding Ayg1-like hydrolase play a role in A. oryzae conidial pigment biosynthesis. However, the involvement of these two genes in conidial pigmentation remains unclear. In this study, we tested this hypothesis by assessing the conidial colors of both disruption and overexpression mutants to verify whether AO090102000546 and AO090005000332 were associated with the conidial pigmentation. Here, observation of single, double, and triple disruptants of these three genes suggested that conidial pigments were synthesized by two laccase genes, AO090011000755 and AO090102000546, whereas Ayg1-like hydrolase gene AO090005000332 was proven to have no obvious association with the synthesis. This was corroborated by observing the phenotype of each overexpression mutant. Interestingly, AO090005000332 overexpression mutant produced smoky yellow-green conidia, different from the wild-type strain. Thus, the AO090005000332-encoded protein is likely to maintain the enzymatic activity. However, the expression level was observed to be one-third of that of AO090102000546 and one-seventh of that of AO090011000755. Consequently, apparent lack of obvious contribution of AO090005000332 to conidial pigmentation could be attributed to its low expression level. Expression analysis indicated similar profiles in several wild-type strains.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2001

Crystallization and initial X-ray diffraction analysis of human pyruvate dehydrogenase

Human pyruvate dehydrogenase (E1) is a component enzyme of the pyruvate dehydrogenase complex. The enzyme catalyzes the irreversible decarboxylation of pyruvic acid and the rate-limiting reductive acetylation of the lipoyl moiety linked to the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. E1 is an alpha(2)beta(2) tetramer ( approximately 154 kDa). Crystals of this recombinant enzyme have been grown in polyethylene glycol 3350 using a vapor-diffusion method at 295 K. The crystals are characterized as orthorhombic, space group P2(1)2(1)2(1), with unit-cell parameters a = 64.2, b = 126.9, c = 190.2 A. Crystals diffracted to a minimum d spacing of 2.5 A. The asymmetric unit contains one alpha(2)beta(2) tetrameric E1 assembly; self-rotation function analysis showed a pseudo-twofold symmetry relating the two alphabeta dimers.

Pyruvate Dehydrogenase Complex/chemistry↗
DOE OSTI2024

A Structurally Diverse Compound Screening Library to Identify Substrates for Diamine, Polyamine, and Related Acetyltransferases

Spermidine/spermine N-acetyltransferases (SSATs) and other types of polyamine acetyltransferases (PAATs) acetylate diamines and/or polyamines. These enzymes are evolutionarily related and belong to the Gcn5-related N-acetyltransferase (GNAT) superfamily, yet we lack a fundamental understanding of their substrate specificity and/or promiscuity toward different compounds. Many of these enzymes are known or are predicted to acetylate polyamines, but in the cell there are other types of compounds that contain moieties derived from polyamines that may be the native substrates for these enzymes. To learn more about the identity of substrates that are acetylated, we selected and screened 17 different GNAT enzymes for activity toward a set of structurally diverse compounds that contained different types of amine moieties (e.g., aminopropyl, aminobutyl, etc.). These compounds included diamines, triamines, and polyamines containing primary amino groups, and they had structural diversity with variation of the chain length and presence or absence of internal amino groups and other functional groups. We found 12 of the 17 enzymes acetylated at least one of the compounds. Some enzymes were selective toward acetylating only one compound while others exhibited substrate promiscuity toward numerous compounds. Our experimental results ultimately allowed us to pinpoint specific substrates that could be further investigated to more fully understand substrate specificity versus promiscuity of GNAT enzymes and the role of acetylated small molecules in cells.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Plant root associated chitinases: structures and functions

Chitinases degrade chitin, a linear homopolymer of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues found in the cell walls of fungi and the exoskeletons of arthropods. They are secreted by the roots into the rhizosphere, a complex and dynamic environment where intense nutrient exchange occurs between plants and microbes. Here we modeled, expressed, purified, and characterized Zea mays and Oryza sativa root chitinases, and the chitinase of a symbiotic bacterium, Chitinophaga oryzae 1303 for their activities with chitin, di-, tri-, and tetra-saccharides and Aspergillus niger, with the goal of determining their role(s) in the rhizosphere and better understanding the molecular mechanisms underlying plant-microbe interactions. We show that Zea mays basic endochitinase (ZmChi19A) and Oryza sativa chitinase (OsChi19A) are from the GH19 chitinase family. The Chitinophaga oryzae 1303 chitinase (CspCh18A) belongs to the GH18 family. The three enzymes have similar apparent K M values of (20-40 µM) for the substrate 4-MU-GlcNAc 3 . They vary in their pH and temperature optima with OsChi19A activity optimal between pH 5–7 and 30–40°C while ZmChi19A and CspCh18A activities were optimal at pH 7-9 and 50–60°C. Modeling and site-directed mutation of ZmChi19A identified the catalytic cleft and the active residues E147 and E169 strategically positioned at ~8.6Å from each other in the folded protein. Cleavage of 4-MU-GlcNAc 3 was unaffected by the absence of the CBD but diminished in the absence of the flexible C-terminal domain. However, unlike for the soluble substrate, the CBD and the newly identified flexible C-terminal domain were vital for inhibiting Aspergillus niger growth. The results are consistent with the involvement of the plant chitinases in defense against pathogens like fungi that have chitin exoskeletons. In summary, we have characterized the functional features and structural domains necessary for the activity of two plant root chitinases that are believed to be involved in plant defense and a bacterial chitinase that, along with the plant chitinases, may participate in nutrient recycling in the rhizosphere.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Engineering the hyperthermophilic archaeon Pyrococcus furiosus for 1-propanol production

ABSTRACT Society relies heavily on chemicals traditionally produced through the refinement of fossil fuels. The conversion of renewable biomass to value-added chemicals by microbes, particularly hyperthermophiles (T opt ≥80°C), offers a renewable alternative to this traditional approach. Herein, we describe the engineering of the hyperthermophilic archaeon Pyrococcus furiosus , which grows optimally (T opt ) at 100°C, for the conversion of sugar to 1-propanol. This was accomplished by constructing a hybrid metabolic pathway consisting of two native and seven heterologously produced enzymes to convert acetyl-CoA from carbohydrate metabolism to 1-propanol. A total of eleven foreign genes from two other organisms were utilized, one from the thermophilic bacterium Thermoanaerobacter sp. strain X514 and 10 from the thermoacidophilic archaeon Metallosphaera sedula, both of which grow optimally near 70°C. The recombinant P. furiosus strain produced 1-propanol at similar concentrations (up to ~1 mM) when incubated at 75°C to activate the gene products of Thermoanaerobacter sp. strain X514 and M. sedula and by initially incubating at 95°C for P. furiosus growth and then subsequently returning to 75°C to promote 1-propanol formation. Note that 1-propanol was not produced if the culture was grown only at 95°C. This work has the potential for future optimization through harnessing the genome-scale metabolic model of P. furiosus that was used herein to identify engineering targets to increase 1-propanol titer. IMPORTANCE As petroleum reserves become increasingly strained, the development of renewable alternatives to traditional chemical synthesis becomes more important. In this work, a high-temperature biological system for sugar to 1-propanol conversion was demonstrated by metabolic engineering of the hyperthermophilic archaeon Pyrococcus furiosus (T opt 100°C). The engineered strain produced 1-propanol by temperature shifting from 75°C to 95°C and then back to 75°C to accommodate the temperature ranges for native and foreign proteins associated with 1-propanol biosynthesis. Genome-scale metabolic modeling informed the carbon and reductant flux in the system, identified potential factors limiting 1-propanol production, and revealed potential optimization targets.

O'Quinn, Hailey C. (ORCID:0009000274865755)↗