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Biotechnology For Biofuels and... Apr 2023Medium-chain fatty acids are molecules with applications in different industries and with growing demand. However, the current methods for their extraction are not...
BACKGROUND
Medium-chain fatty acids are molecules with applications in different industries and with growing demand. However, the current methods for their extraction are not environmentally sustainable. The reverse β-oxidation pathway is an energy-efficient pathway that produces medium-chain fatty acids in microorganisms, and its use in Saccharomyces cerevisiae, a broadly used industrial microorganism, is desired. However, the application of this pathway in this organism has so far either led to low titers or to the predominant production of short-chain fatty acids.
RESULTS
We genetically engineered Saccharomyces cerevisiae to produce the medium-chain fatty acids hexanoic and octanoic acid using novel variants of the reverse β-oxidation pathway. We first knocked out glycerolphosphate dehydrogenase GPD2 in an alcohol dehydrogenases knock-out strain (△adh1-5) to increase the NADH availability for the pathway, which significantly increased the production of butyric acid (78 mg/L) and hexanoic acid (2 mg/L) when the pathway was expressed from a plasmid with BktB as thiolase. Then, we tested different enzymes for the subsequent pathway reactions: the 3-hydroxyacyl-CoA dehydrogenase PaaH1 increased hexanoic acid production to 33 mg/L, and the expression of enoyl-CoA hydratases Crt2 or Ech was critical to producing octanoic acid, reaching titers of 40 mg/L in both cases. In all cases, Ter from Treponema denticola was the preferred trans-enoyl-CoA reductase. The titers of hexanoic acid and octanoic acid were further increased to almost 75 mg/L and 60 mg/L, respectively, when the pathway expression cassette was integrated into the genome and the fermentation was performed in a highly buffered YPD medium. We also co-expressed a butyryl-CoA pathway variant to increase the butyryl-CoA pool and support the chain extension. However, this mainly increased the titers of butyric acid and only slightly increased that of hexanoic acid. Finally, we also tested the deletion of two potential medium-chain acyl-CoA depleting reactions catalyzed by the thioesterase Tes1 and the medium-chain fatty acyl CoA synthase Faa2. However, their deletion did not affect the production titers.
CONCLUSIONS
By engineering the NADH metabolism and testing different reverse β-oxidation pathway variants, we extended the product spectrum and obtained the highest titers of octanoic acid and hexanoic acid reported in S. cerevisiae. Product toxicity and enzyme specificity must be addressed for the industrial application of the pathway in this organism.
PubMed: 37101299
DOI: 10.1186/s13068-023-02317-z -
Biotechnology and Bioengineering Jul 2017Biorenewable chemicals such as short and medium chain fatty acids enable functional or direct substitution of petroleum-derived building blocks, allowing reduction of...
Biorenewable chemicals such as short and medium chain fatty acids enable functional or direct substitution of petroleum-derived building blocks, allowing reduction of anthropogenic greenhouse gases while meeting market needs of high-demand products like aliphatic alcohols and alpha olefins. However, producing these fatty acids in microorganisms can be challenging due to toxicity issues. Octanoic acid (C8) can disrupt the integrity of the cell membrane in yeast, and exogenous supplementation of oleic acid has been shown to help alleviate this. We recently engineered the Saccharomyces cerevisiae enzyme acetyl-CoA carboxylase by replacing serine residue 1157 with alanine to prevent deactivation by phosphorylation. Expression of Acc1 in S. cerevisiae resulted in an increase in total fatty acid production, with the largest increase for oleic acid. In this study, we evaluated the effect of this modified lipid profile on C8 toxicity to the yeast. Expression of Acc1 in S. cerevisiae BY4741 increased the percentage of oleic acid 3.1- and 1.6-fold in the absence and presence of octanoic acid challenge, respectively. Following exposure to 0.9 mM of C8 for 24 h, the engineered yeast had a 10-fold higher cell density relative to the baseline strain. Moreover, overexpressing Acc1 allowed survival at C8 concentrations that were lethal for the baseline strain. This marked reduction of toxicity was shown to be due to higher membrane integrity as an 11-fold decrease in leakage of intracellular magnesium was observed. Due to the increase in oleic acid, this approach has the potential to reduce toxicity of other valuable bioproducts such as shorter chain aliphatic acids and alcohols and other membrane stressors. In an initial screen, increased resistance to n-butanol, 2-propanol, and hexanoic acid was demonstrated with cell densities 3.2-, 1.8-, and 29-fold higher than the baseline strain, respectively. Biotechnol. Bioeng. 2017;114: 1531-1538. © 2017 Wiley Periodicals, Inc.
Topics: Acetyl-CoA Carboxylase; Caprylates; Cell Survival; Fatty Acids; Genetic Enhancement; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 28294288
DOI: 10.1002/bit.26288 -
Scientific Reports Mar 2021Octanoic acid is a medium-chained saturated fatty acid found abundantly in the ketogenic dietary supplements containing medium chained triglycerides (MCT) along with...
Octanoic acid is a medium-chained saturated fatty acid found abundantly in the ketogenic dietary supplements containing medium chained triglycerides (MCT) along with decanoic acid. The MCT ketogenic diet is commonly consumed for weight loss but has also showcased neuroprotective potential against neurodegenerative disorders. However, recent clinical findings have reported a critical disadvantage with the long-term consumption of ketogenic diet i.e. bone loss. The following study was employed to investigate whether the two major components of MCT diet also possess bone loss potential as observed with classical ketogenic diet. Swiss albino mice aged between 10 and 12 weeks, were divided into 3 treatment groups that were administered with oral suspensions of octanoic acid, decanoic acid and a combination of both for 4 weeks. Bone specific markers, microarchitectural parameters, using micro computed tomography, and biomechanical strength were analyzed. Remarkably deleterious alterations in the trabecular bone microarchitecture, and on bone markers were observed in the octanoic acid treated groups. Our results suggest significant negative effects on bone health by octanoic acid. These findings require further investigation and validation in order to provide significant clinically relevant data to possibly modify dietary composition of the MCT ketogenic diet.
Topics: Animals; Biomechanical Phenomena; Bone Density; Bone Resorption; Cancellous Bone; Caprylates; Decanoic Acids; Diet, High-Protein Low-Carbohydrate; Diet, Ketogenic; Dietary Supplements; Femur; Ketone Bodies; Male; Mice; Neuroprotective Agents; Osteoclasts; Random Allocation; Tibia; Triglycerides
PubMed: 33772066
DOI: 10.1038/s41598-021-86468-9 -
Food and Chemical Toxicology : An... Apr 2020
Review
Topics: Animals; Caprylates; Humans; Perfume; Registries; Risk Assessment; Toxicity Tests; Volatile Organic Compounds
PubMed: 32199972
DOI: 10.1016/j.fct.2020.111271 -
Frontiers in Neuroscience 2020Medium-chain fatty acids (MCFA) are dietary components with a chain length ranging from 6 to 12 carbon atoms. MCFA can cross the blood-brain barrier and in the brain can...
Medium-chain fatty acids (MCFA) are dietary components with a chain length ranging from 6 to 12 carbon atoms. MCFA can cross the blood-brain barrier and in the brain can be oxidized through mitochondrial β-oxidation. As components of ketogenic diets, MCFA have demonstrated beneficial effects on different brain diseases, such as traumatic brain injury, Alzheimer's disease, drug-resistant epilepsy, diabetes, and cancer. Despite the interest in MCFA effects, not much information is available about MCFA metabolism in the brain. In this study, with a gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach, coupled with multivariate data analyses, we followed the metabolic changes of U87MG glioblastoma cells after the addition of octanoic (C8), or decanoic (C10) acids for 24 h. Our analysis highlighted significant differences in the metabolism of U87MG cells after the addition of C8 or C10 and identified several metabolites whose amount changed between the two groups of treated cells. Overall, metabolic pathway analyses suggested the citric acid cycle, Warburg effect, glutamine/glutamate metabolism, and ketone body metabolism as pathways influenced by C8 or C10 addition to U87MG cells. Our data demonstrated that, while C8 affected mitochondrial metabolism resulting in increased ketone body production, C10 mainly influenced cytosolic pathways by stimulating fatty acid synthesis. Moreover, glutamine might be the main substrate to support fatty acids synthesis in C10-treated cells. In conclusion, we identified a metabolic signature associated with C8 or C10 addition to U87MG cells that can be used to decipher metabolic responses of glioblastoma cells to MCFA treatment.
PubMed: 32792906
DOI: 10.3389/fnins.2020.00783 -
Chemistry (Weinheim An Der Bergstrasse,... Jan 2021The chemical background of olfactory perception has been subject of intensive research, but no available model can fully explain the sense of smell. There are also...
The chemical background of olfactory perception has been subject of intensive research, but no available model can fully explain the sense of smell. There are also inconsistent results on the role of the isotopology of molecules. In experiments with human subjects it was found that the isotope effect is weak with acetone and D -acetone. In contrast, clear differences were observed in the perception of octanoic acid and D -octanoic acid. Furthermore, a trained sniffer dog was initially able to distinguish between these isotopologues of octanoic acid. In chromatographic measurements, the respective deuterated molecule showed weaker interaction with a non-polar liquid phase. Quantum chemical calculations give evidence that deuterated octanoic acid binds more strongly to a model receptor than non-deuterated. In contrast, the binding of the non-deuterated molecule is stronger with acetone. The isotope effect is calculated in the framework of statistical mechanics. It results from a complicated interplay between various thermostatistical contributions to the non-covalent free binding energies and it turns out to be very molecule-specific. The vibrational terms including non-classical zero-point energies play about the same role as rotational/translational contributions and are larger than bond length effects for the differential isotope perception of odor for which general rules cannot be derived.
Topics: Acetone; Animals; Caprylates; Deuterium; Dogs; Odorants; Olfactory Perception; Organic Chemicals; Smell; Working Dogs
PubMed: 33058253
DOI: 10.1002/chem.202003754 -
Journal of Biotechnology Jul 2016The present proof-of-concept study reports the construction of a whole-cell biocatalyst for the de novo production of ω-hydroxy octanoic acid. This was achieved by...
The present proof-of-concept study reports the construction of a whole-cell biocatalyst for the de novo production of ω-hydroxy octanoic acid. This was achieved by hijacking the natural fatty acid cycle and subsequent hydroxylation using a specific monooxygenase without the need for the additional feed of alkene-like precursors. For this, we used the model organism Escherichia coli and increased primarily the release of the octanoic acid precursors by overexpressing the plant thioesterase FatB2 from Cuphea hookeriana in a β-oxidation deficient strain, which lead to the production of 2.32mM (8.38mggcww(-1)) octanoic acid in 24h. In order to produce the corresponding ω-hydroxy derivative, we additionally expressed the engineered self-sufficient monooxygenase fusion protein CYP153AMaq(G307A)-CPRBM3 within the octanoic acid producing strain. With this, we finally produced 234μM (0.95mggcww(-1)) ω-hydroxy octanoic acid in a 20h fed-batch set-up.
Topics: Bioreactors; Caprylates; Cuphea; Escherichia coli; Plant Proteins; Thiolester Hydrolases
PubMed: 27184430
DOI: 10.1016/j.jbiotec.2016.05.017 -
Applied Microbiology and Biotechnology Dec 2016Octanoic acid (OA) precipitation has long been used in protein purification. Recently, we reported a new cell culture clarification method for immunoglobulin G (IgG)...
Octanoic acid (OA) precipitation has long been used in protein purification. Recently, we reported a new cell culture clarification method for immunoglobulin G (IgG) purification, employing an advance elimination of chromatin heteroaggregates with a hybrid OA-solid phase system. This treatment reduced DNA more than 3 logs, histone below the detection limit (LOD), and non-histone host cell proteins (nh-HCP) by 90 % while conserving more than 90 % of the IgG monomer. In this study, we further investigated the conservation of IgG monomer and antibody light chain (LC) to the addition of OA/OA-solid phase complex, with or without histone and DNA in different combinations. The results showed that highly basic histone protein was the prime target in OA/OA-solid phase precipitation system for IgG purification, and the selective conservation of IgG monomer in this system was histone dependent. Our findings partially support the idea that OA works by sticking to electropositive hydrophobic domains on proteins, reducing their solubility, and causing them to agglomerate into large particles that precipitate from solution. Our findings also provide a new perspective for IgG purification and emphasize the necessity to re-examine the roles of various host contaminants in IgG purification.
Topics: Caprylates; Chemical Precipitation; Chromatography; Heterochromatin; Histones; Immunoglobulin G; Immunologic Factors; Recombinant Proteins
PubMed: 27418358
DOI: 10.1007/s00253-016-7719-x -
Frontiers in Bioengineering and... 2021Production of medium chain-length poly(3-hydroxyalkanoates) [PHA] polymers with tightly defined compositions is an important area of research to expand the application...
Production of medium chain-length poly(3-hydroxyalkanoates) [PHA] polymers with tightly defined compositions is an important area of research to expand the application and improve the properties of these promising biobased and biodegradable materials. PHA polymers with homopolymeric or defined compositions exhibit attractive material properties such as increased flexibility and elasticity relative to poly(3-hydroxybutyrate) [PHB]; however, these polymers are difficult to biosynthesize in native PHA-producing organisms, and there is a paucity of research toward developing high-density cultivation methods while retaining compositional control. In this study, we developed and optimized a fed-batch fermentation process in a stirred tank reactor, beginning with the biosynthesis of poly(3-hydroxydecanoate) [PHD] from decanoic acid by β-oxidation deficient recombinant LSBJ using glucose as a co-substrate solely for growth. Bacteria were cultured in two stages, a biomass accumulation stage (37°C, pH 7.0) with glucose as the primary carbon source and a PHA biosynthesis stage (30°C, pH 8.0) with co-feeding of glucose and a fatty acid. Through iterative optimizations of semi-defined media composition and glucose feed rate, 6.0 g of decanoic acid was converted to PHD with an 87.5% molar yield (4.54 g L). Stepwise increases in the amount of decanoic acid fed during the fermentation correlated with an increase in PHD, resulting in a final decanoic acid feed of 25 g converted to PHD at a yield of 89.4% (20.1 g L, 0.42 g L h), at which point foaming became uncontrollable. Hexanoic acid, octanoic acid, 10-undecenoic acid, and 10-bromodecanoic acid were all individually supplemented at 20 g each and successfully polymerized with yields ranging from 66.8 to 99.0% (9.24 to 18.2 g L). Using this bioreactor strategy, co-fatty acid feeds of octanoic acid/decanoic acid and octanoic acid/10-azidodecanoic acid (8:2 mol ratio each) resulted in the production of their respective copolymers at nearly the same ratio and at high yield, demonstrating that these methods can be used to control PHA copolymer composition.
PubMed: 33718339
DOI: 10.3389/fbioe.2021.618259 -
Brain Sciences Oct 2017This review focuses on biomarkers associated with the outcome of traumatic brain injury (TBI) patients, such as caspase-3; total antioxidant capacity; melatonin; S100B... (Review)
Review
This review focuses on biomarkers associated with the outcome of traumatic brain injury (TBI) patients, such as caspase-3; total antioxidant capacity; melatonin; S100B protein; glial fibrillary acidic protein (GFAP); glutamate; lactate; brain-derived neurotrophic factor (BDNF); substance P; neuron-specific enolase (NSE); ubiquitin carboxy-terminal hydrolase L-1 (UCH-L1); tau; decanoic acid; and octanoic acid.
PubMed: 29076989
DOI: 10.3390/brainsci7110142