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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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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Large-scale Genetic Characterization of a Model Sulfate-Reducing Bacterium

ABSTRACTSulfate-reducing bacteria (SRB) are obligate anaerobes that can couple their growth to the reduction of sulfate. Despite the importance of SRB to global nutrient cycles and their damage to the petroleum industry, our molecular understanding of their physiology remains limited. To systematically provide new insights into SRB biology, we generated a randomly barcoded transposon mutant library in the model SRB Desulfovibrio vulgaris Hildenborough (DvH) and used this genome-wide resource to assay the importance of its genes under a range of metabolic and stress conditions. In addition to defining the essential gene set of DvH, we identified a conditional phenotype for 1,137 non-essential genes. Through examination of these conditional phenotypes, we were able to make a number of novel insights into our molecular understanding of DvH, including how this bacterium synthesizes vitamins. For example, we identified DVU0867 as an atypical L-aspartate decarboxylase required for the synthesis of pantothenic acid, provided the first experimental evidence that biotin synthesis in DvH occurs via a specialized acyl carrier protein and without methyl esters, and demonstrated that the uncharacterized dehydrogenase DVU0826:DVU0827 is necessary for the synthesis of pyridoxal phosphate. In addition, we used the mutant fitness data to identify genes involved in the assimilation of diverse nitrogen sources, and gained insights into the mechanism of inhibition of chlorate and molybdate. Our large-scale fitness dataset and RB-TnSeq mutant library are community-wide resources that can be used to generate further testable hypotheses into the gene functions of this environmentally and industrially important group of bacteria.

Trotter, Valentine V↗
DOE OSTI2023

Large-scale genetic characterization of the model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough

Sulfate-reducing bacteria (SRB) are obligate anaerobes that can couple their growth to the reduction of sulfate. Despite the importance of SRB to global nutrient cycles and their damage to the petroleum industry, our molecular understanding of their physiology remains limited. To systematically provide new insights into SRB biology, we generated a randomly barcoded transposon mutant library in the model SRB Desulfovibrio vulgaris Hildenborough (DvH) and used this genome-wide resource to assay the importance of its genes under a range of metabolic and stress conditions. In addition to defining the essential gene set of DvH, we identified a conditional phenotype for 1,137 non-essential genes. Through examination of these conditional phenotypes, we were able to make a number of novel insights into our molecular understanding of DvH, including how this bacterium synthesizes vitamins. For example, we identified DVU0867 as an atypical L-aspartate decarboxylase required for the synthesis of pantothenic acid, provided the first experimental evidence that biotin synthesis in DvH occurs via a specialized acyl carrier protein and without methyl esters, and demonstrated that the uncharacterized dehydrogenase DVU0826:DVU0827 is necessary for the synthesis of pyridoxal phosphate. In addition, we used the mutant fitness data to identify genes involved in the assimilation of diverse nitrogen sources and gained insights into the mechanism of inhibition of chlorate and molybdate. Our large-scale fitness dataset and RB-TnSeq mutant library are community-wide resources that can be used to generate further testable hypotheses into the gene functions of this environmentally and industrially important group of bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Treatment of cholinergic‐induced status epilepticus with polytherapy targeting GABA and glutamate receptors

Abstract Despite new antiseizure medications, the development of cholinergic‐induced refractory status epilepticus (RSE) continues to be a therapeutic challenge as pharmacoresistance to benzodiazepines and other antiseizure medications quickly develops. Studies conducted by Epilepsia . 2005;46:142 demonstrated that the initiation and maintenance of cholinergic‐induced RSE are associated with trafficking and inactivation of gamma‐aminobutyric acid A receptors (GABA A R) thought to contribute to the development of benzodiazepine pharmacoresistance. In addition, Dr. Wasterlain's laboratory reported that increased N‐methyl‐ d ‐aspartate receptors (NMDAR) and alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors (AMPAR) contribute to enhanced glutamatergic excitation ( Neurobiol Dis . 2013;54:225; Epilepsia . 2013;54:78). Thus, Dr. Wasterlain postulated that targeting both maladaptive responses of reduced inhibition and increased excitation that is associated with cholinergic‐induced RSE should improve therapeutic outcome. We currently review studies in several animal models of cholinergic‐induced RSE that demonstrate that benzodiazepine monotherapy has reduced efficacy when treatment is delayed and that polytherapy with drugs that include a benzodiazepine (eg midazolam and diazepam) to counter loss of inhibition, concurrent with an NMDA antagonist (eg ketamine) to reduce excitation provide improved efficacy. Improved efficacy with polytherapy against cholinergic‐induced seizure is demonstrated by reduction in (1) seizure severity, (2) epileptogenesis, and (3) neurodegeneration compared with monotherapy. Animal models reviewed include pilocarpine‐induced seizure in rats, organophosphorus nerve agent (OPNA)‐induced seizure in rats, and OPNA‐induced seizure in two mouse models: (1) carboxylesterase knockout (Es1 −/− ) mice which, similarly to humans, lack plasma carboxylesterase and (2) human acetylcholinesterase knock‐in carboxylesterase knockout (KIKO) mice. We also review studies showing that supplementing midazolam and ketamine with a third antiseizure medication (valproate or phenobarbital) that targets a nonbenzodiazepine site rapidly terminates RSE and provides further protection against cholinergic‐induced SE. Finally, we review studies on the benefits of simultaneous compared with sequential drug treatments and the clinical implications that lead us to predict improved efficacy of early combination drug therapies. The data generated from seminal rodent studies of efficacious treatment of cholinergic‐induced RSE conducted under Dr. Wasterlain's guidance suggest that future clinical trials should treat the inadequate inhibition and temper the excess excitation that characterize RSE and that early combination therapies may provide improved outcome over benzodiazepine monotherapy.

59 BASIC BIOLOGICAL SCIENCES↗
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 OSTI2026

High yield production of 3-hydroxypropionic acid using Issatchenkia orientalis

Biomanufacturing provides a more sustainable alternative to fossil-based chemical manufacturing. 3-Hydroxypropionic acid (3HP) is a top Department of Energy value-added chemical and precursor to bioplastics, yet cost-effective microbial production remains elusive. Here, we establish the acid-tolerant yeast Issatchenkia orientalis as a robust host for low-pH 3HP biosynthesis. Genome-scale modeling identifies the β-alanine pathway as optimal, offering the highest theoretical yield and lowest oxygen requirement. Thermodynamic analysis confirms its favorability under acidic conditions. Using sequence similarity network analysis, we discover highly active aspartate 1-decarboxylase (PAND), β-alanine-pyruvate aminotransferase (BAPAT), and 3HP dehydrogenase (YDFG), which significantly improve the pathway efficiency. Next, to further elevate the production, pathway optimization through multi-copy PAND integration, byproduct elimination (knockouts of pyruvate decarboxylase and glycerol-3-phosphate dehydrogenase), and reinforcement of aspartate flux by overexpression of pyruvate carboxylase and aspartate amino transferase improves the titer to 29 g/L in shake flasks. Fed-batch fermentation at pH 4 with low-cost corn steep liquor medium further increases the production to 92 g/L with 0.7 g/g yield and 0.55 g/L/h productivity. Techno-economic analysis indicates that such performance could potentially enable a financially viable process for sustainable acrylic acid production. This work establishes I. orientalis as a next-generation platform for cost-effective 3HP production and paves the way toward industrial commercialization.

Biotechnology↗
DOE OSTI2022

Regulation of L - and D -Aspartate Transport and Metabolism in Acinetobacter baylyi ADP1

Here, the regulated uptake and consumption of d-amino acids by bacteria remain largely unexplored, despite the physiological importance of these compounds. Unlike other characterized bacteria, such as Escherichia coli, which utilizes only l-Asp, Acinetobacter baylyi ADP1 can consume both d-Asp and l-Asp as the sole carbon or nitrogen source. As described here, two LysR-type transcriptional regulators (LTTRs), DarR and AalR, control d- and l-Asp metabolism in strain ADP1. Heterologous expression of A. baylyi proteins enabled E. coli to use d-Asp as the carbon source when either of two transporters (AspT or AspY) and a racemase (RacD) were coexpressed. A third transporter, designated AspS, was also discovered to transport Asp in ADP1. DarR and/or AalR controlled the transcription of aspT, aspY, racD, and aspA (which encodes aspartate ammonia lyase). Conserved residues in the N-terminal DNA-binding domains of both regulators likely enable them to recognize the same DNA consensus sequence (ATGC-N7-GCAT) in several operator-promoter regions. In strains lacking AalR, suppressor mutations revealed a role for the ClpAP protease in Asp metabolism. In the absence of the ClpA component of this protease, DarR can compensate for the loss of AalR. ADP1 consumed l- and d-Asn and l-Glu, but not d-Glu, as the sole carbon or nitrogen source using interrelated pathways.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Channelrhodopsin C1C2: Photocycle kinetics and interactions near the central gate

Channelrhodopsins (ChR) are light-sensitive cation channels used in optogenetics, a technique that applies light to control cells (e.g., neurons) that have been modified genetically to express those channels. Although mutations are known to affect pore kinetics, little is known about how mutations induce changes at the molecular scale. To address this issue, we first measured channel opening and closing rates of a ChR chimera (C1C2) and selected variants (N297D, N297V, and V125L). Then, we used atomistic simulations to correlate those rates with changes in pore structure, hydration, and chemical interactions among key gating residues of C1C2 in both closed and open states. Overall, the experimental results show that C1C2 and its mutants do not behave like ChR2 or its analogous variants, except V125L, making C1C2 a unique channel. Furthermore, our atomistic simulations confirmed that opening of the channel and initial hydration of the gating regions between helices I, II, III, and VII of the channel occurs with 1) the presence of 13-cis retinal; 2) deprotonation of a glutamic acid gating residue, E129; and 3) subsequent weakening of the central gate hydrogen bond between the same glutamic acid E129 and asparagine N297 in the central region of the pore. Also, an aspartate (D292) is the unambiguous primary proton acceptor for the retinal Schiff base in the hydrated channel.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Postbiotic Gamma-Aminobutyric Acid and Camel Milk Intervention as Innovative Trends Against Hyperglycemia and Hyperlipidemia in Streptozotocin-Induced C57BL/6J Diabetic Mice

Diabetes is a serious disease that threatens human health worldwide. The study hypothesis is to investigate the novel trends that may aid in the prevention of diabetic complications. Camel milk was presented as traditional functional food, and Lactobacillus brevis KLDS 1.0727 and KLDS 1.0373 strains were shown to synthesize postbiotic Gamma-aminobutyric acid as a potential food additive, which can therapeutically intervene against hyperglycemia and hyperlipidemia in streptozotocin-induced C 57 BL/6J mice. During a four-week timeframe, body weight and postprandial blood glucose levels were monitored. Post-euthanasia, blood plasma was obtained to investigate hyperlipidemia, insulin concentrations, liver, and renal functions. The liver, pancreas, kidney, and spleen underwent histopathological examinations. The results demonstrated that KLDS 1.0727 and KLDS 1.0373 ( LAC S1 , LAC S2 ) and camel milk treatments all had a significant influence on hypoglycemic activity, as evidenced by reduced postprandial blood glucose levels. LAC S1 , LAC S2 , and camel milk therapy significantly reduced blood hypolipidemic, and some liver enzymes such as (alanine aminotransferase and aspartate transaminase) levels. Therefore, we recommend consuming camel milk regularly and expanding its use with fermented foods containing L. brevis , one of the probiotics capable of producing gamma-aminobutyric acid (GABA) as future food additives that can improve human health and reduce the prevalence of several diseases disorders.

Abdelazez, Amro↗
DOE OSTI2024

Reduced ribonucleotide reductase RRM2 subunit expression increases DNA damage and mitochondria dysfunction in woody breast chickens

Abstract OBJECTIVE The aim of this study was to investigate the roles of ribonucleotide reductase subunit M2 (RRM2; subunit of ribonucleotide reductase) in severe woody breast (WB) and normal breast muscles. ANIMALS 40 8-week-old male Ross-708 broiler chickens. METHODS Quantitative PCR was performed to determine gene expression, and commercial ELISA/assay kits were used to obtain several enzymatic activities. RESULTS Results showed that RRM2 activity (P= .0002) and RRM2 (P= .05) and hydroxymethylbilane synthase expression (impaired oxygen transport and metabolism,P= .002) were reduced in WB, while caveolin-3 (defected membrane integrity,P= .09), endoglin (increased fibrosis,P= .06), and secreted protein acidic rich in cysteine (metabolic dysregulation,P= .09) expression tended to increase in WB. WB tended to have increased levels of homocysteine (P= .06), aspartate aminotransferase mitochondria (P= .02), pyruvate kinase (P= .04), DNA damage (P= .06), creatine kinase (P= .05), and triglyceride (P= .002) but decreased ATPase activity (P= .01), all indicating mitochondria dysfunction and tissue damage. CLINICAL RELEVANCE In this study, differences in various enzyme activities and increased DNA damage suggest that RRM2-mediated mitochondrial abnormalities may play a role in WB myopathy.

Veterinary Sciences↗
DOE OSTI2025

Conformational free energy landscape of a glutamate transporter and microscopic details of its transport mechanism

Removing glutamate from the synaptic cleft is vital for proper function of the brain. Excitatory amino acid transporters mediate this process by uptaking the neurotransmitter from the synaptic cleft back to the cell after its release. The archaeal homolog, Glt Ph , an aspartate transporter fromPyrococcus horikoshii, presents the best structurally characterized model for this family of transporters. In order to transport, Glt Ph undergoes elevator-like conformational changes between inward-facing (IF) and outward-facing (OF) states. Here, we characterize, at an atomic level, the OF⇌IF transition of Glt Ph in differentapo/bound states using a combination of ensemble-based enhanced sampling techniques, employing more than two thousand of coupled simulation replicas of membrane-embedded Glt Ph . The resulting free-energy profiles portray the transition ofapo/bound states as a complex four-stage process, while sodium binding alone locks the structure in one of its states. Along the transition, the transport domain (TD) disengages from the scaffold domain (SD), allowing it to move as a piston sliding vertically with respect to the membrane during the elevator-like motion of TD. Lipid interactions with residues comprising the SD–TD interface directly influence the large-scale conformational changes and, consequently, the energetics of transport. Structural intermediates formed during the transition leak water molecules and may correlate to the uncoupled Cl − ion conductance observed experimentally in both prokaryotic and mammalian glutamate transporters. Mechanistic insights obtained from our study provide a structural framework for better development of therapeutic for neurological disorders.

Science & Technology - Other Topics↗
DOE OSTI2020

Aspartate Residues in a Forisome-Forming SEO Protein Are Critical for Protein Body Assembly and Ca 2+ Responsiveness

Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca 2+ -controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca 2+ -dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein–protein interaction and Ca 2+ -binding motifs. Here we selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca 2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast ( Saccharomyces cerevisiae ) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca 2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca 2+ -controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca 2+ -dependent contractility.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Precision Labeling of Native Antibodies with Lock Coupling

The formation of stable protein complexes enables much of biotechnology, but even high-affinity complexes can dissociate, limiting potential applications in biomaterials, bioimaging, nanomedicine, and other protein-based technologies. Here, in this study, we describe lock coupling, a simple and selective one-step reaction between interfacial lysine and glutamate or aspartate side chains to form stable isopeptide bonds and be used for the precise labeling of native antibodies. We identify conditions in which short-lived activated esters formed by the aqueous carbodiimide EDC promote isopeptide bond formation specifically at preassociated amine-acid pairs. Indiscriminate cross-linking is minimized by formation of protein complexes before addition of catalyst, use of acidic pH to suppress exposed Lys reactivity, and limiting the aqueous stability of activated esters. For native antibody (Ab) labeling, we show that the small IgG-binding protein GB1 can be covalently attached to the Ab Fc domain and that introduction of Cys into GB1 loops allows for facile conjugation of fluorophores, micelles, or inorganic nanocrystals for imaging in live cells and animals. By varying Cys substituents and protein stoichiometry, a defined number of probes can be uniformly attached without the need for extensive purification. In live-cell confocal microscopy, labeled GB1 serves as a stable replacement for secondary Abs, enabling simple multicolor immunostaining and imaging. Lock coupling requires just a single reagent in aqueous buffer and leverages both the innate ability of proteins to form high-affinity complexes and the widespread presence of Lys-Glu/Asp pairs at their interfaces, with the potential for precision synthesis of protein-based probes for imaging, biomaterials, biophysics, and medicine.

antibody↗
DOE OSTI2023

Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species

Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. In this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol -1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol -1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol -1 sugars and resulted in a final titer of 36.0 g/L 3-HP. The results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane.

09 BIOMASS FUELS↗
DOE OSTI2022

Corynebacterium glutamicum as an Efficient Omnivorous Microbial Host for the Bioconversion of Lignocellulosic Biomass

Corynebacterium glutamicum has been successfully employed for the industrial production of amino acids and other bioproducts, partially due to its native ability to utilize a wide range of carbon substrates. We demonstrated C. glutamicum as an efficient microbial host for utilizing diverse carbon substrates present in biomass hydrolysates, such as glucose, arabinose, and xylose, in addition to its natural ability to assimilate lignin-derived aromatics. As a case study to demonstrate its bioproduction capabilities, L-lactate was chosen as the primary fermentation end product along with acetate and succinate. C. glutamicum was found to grow well in different aromatics (benzoic acid, cinnamic acid, vanillic acid, and p-coumaric acid) up to a concentration of 40 mM. Besides, 13 C-fingerprinting confirmed that carbon from aromatics enter the primary metabolism via TCA cycle confirming the presence of β-ketoadipate pathway in C. glutamicum . 13 C-fingerprinting in the presence of both glucose and aromatics also revealed coumarate to be the most preferred aromatic by C. glutamicum contributing 74 and 59% of its carbon for the synthesis of glutamate and aspartate respectively. 13 C-fingerprinting also confirmed the activity of ortho-cleavage pathway, anaplerotic pathway, and cataplerotic pathways. Finally, the engineered C. glutamicum strain grew well in biomass hydrolysate containing pentose and hexose sugars and produced L-lactate at a concentration of 47.9 g/L and a yield of 0.639 g/g from sugars with simultaneous utilization of aromatics. Succinate and acetate co-products were produced at concentrations of 8.9 g/L and 3.2 g/L, respectively. Our findings open the door to valorize all the major carbon components of biomass hydrolysate by using C. glutamicum as a microbial host for biomanufacturing.

13C-fingerprinting↗
DOE OSTI2026

Prediction of non-intuitive metabolic targets with bayesian metabolic control analysis to improve 3-hydroxypropionic acid production in Aspergillus niger

Development of efficient bioconversion processes is limited by the ability to predictably improve metabolic flux. Here we deployed Bayesian Metabolic Control Analysis as a platform to integrate multi-omics data with metabolic modeling and evaluated its ability to predict genetic interventions that improve metabolic flux. Global Metabolomics and proteomics data was collected from 17 Aspergillus niger strains engineered to produce the platform biochemical 3-hydroxypropionic acid from which seven actional genetic interventions were predicted from significant flux control coefficients. Of the suggested genetic interventions, two were present within the intuitively designed strains used for training (malonic semialdehyde dehydrogenase and pyruvate carboxylase) while five predicted targets were present within non-intuitive areas of the metabolic network including 5-formyltetrahydrofolate deformylase and four mitochondrial enzymes, alcohol dehydrogenase, succinyl-CoA ligase, aspartate aminotransferase, and malate dehydrogenase. Six of the targets were validated in the highest performing 3-HP strain used for multi-omics data generation which contained a prior disruption of the highest scoring target malonic semialdehyde dehydrogenase. Predicted directional perturbation of five of the six tested targets significantly improved titer and rate of 3-HP production and two significantly improved yield. The greatest improvements were observed following disruption of the non-intuitive target succinyl-CoA ligase which increased titer by 39% and yield by 29% (to 20.4 g/L 3-HP and 0.31 g 3-HP/g glucose) over the strains used for training. This study demonstrates the utility of Bayesian Metabolic Control Analysis and highlights the ability to predict meaningful genetic targets in unexpected areas of metabolism to improve engineered strains for bioconversion.

3-hydroxypropionic acid↗