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Biotechnology Journal Jun 2023Hydrolysates are used as media supplements although their role is not well characterized. In this study, cottonseed hydrolysates, which contained peptides and galactose...
Hydrolysates are used as media supplements although their role is not well characterized. In this study, cottonseed hydrolysates, which contained peptides and galactose as supplemental substrates, were added to Chinese hamster ovary (CHO) batch cultures, enhancing cell growth, immunoglobulin (IgG) titers, and productivities. Extracellular metabolomics coupled with tandem mass tag (TMT) proteomics revealed metabolic and proteomic changes in cottonseed-supplemented cultures. Shifts in production and consumption dynamics of glucose, glutamine, lactate, pyruvate, serine, glycine, glutamate, and aspartate suggest changes in tricarboxylic acid (TCA) and glycolysis metabolism following hydrolysate inputs. Quantitative proteomics revealed 5521 proteins and numerous changes in relative abundance of proteins related to growth, metabolism, oxidative stress, protein productivity, and apoptosis/cell death at day 5 and day 6. Differential abundance of amino acid transporter proteins and catabolism enzymes such as branched-chain-amino-acid aminotransferase (BCAT)1 and fumarylacetoacetase (FAH) can alter availability and utilization of several amino acids. Also, pathways involved in growth including the polyamine biosynthesis through higher ornithine decarboxylase (ODC1) abundance and hippo signaling were upregulated and downregulated, respectively. Central metabolism rewiring was indicated by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) downregulation, which corresponded with re-uptake of secreted lactate in the cottonseed-supplemented cultures. Overall, cottonseed hydrolysate supplementation modified culture performance by altering cellular activities critical to growth and protein productivity including metabolism, transport, mitosis, transcription, translation, protein processing, and apoptosis. HIGHLIGHTS: Cottonseed hydrolysate, as a medium additive, enhances Chinese hamster ovary (CHO) cell culture performance. Metabolite profiling and tandem mass tag (TMT) proteomics characterize its impact on CHO cells. Rewired nutrient utilization is observed via glycolysis, amino acid, and polyamine metabolism. Hippo signaling pathway impacts cell growth in the presence of cottonseed hydrolysate.
Topics: Cricetinae; Animals; Cricetulus; CHO Cells; Cottonseed Oil; Proteomics; Batch Cell Culture Techniques; Lactic Acid; Pyruvic Acid; Amino Acids; Dietary Supplements; Polyamines
PubMed: 36892270
DOI: 10.1002/biot.202200243 -
Microbial Cell Factories Sep 2018Production of isoprenoids, a large and diverse class of commercially important chemicals, can be achieved through engineering metabolism in microorganisms. Several...
Role of phosphate limitation and pyruvate decarboxylase in rewiring of the metabolic network for increasing flux towards isoprenoid pathway in a TATA binding protein mutant of Saccharomyces cerevisiae.
BACKGROUND
Production of isoprenoids, a large and diverse class of commercially important chemicals, can be achieved through engineering metabolism in microorganisms. Several attempts have been made to reroute metabolic flux towards isoprenoid pathway in yeast. Most approaches have focused on the core isoprenoid pathway as well as on meeting the increased precursors and cofactor requirements. To identify unexplored genetic targets that positively influence the isoprenoid pathway activity, a carotenoid based genetic screen was previously developed and three novel mutants of a global TATA binding protein SPT15 was isolated for heightened isoprenoid flux in Saccharomyces cerevisiae.
RESULTS
In this study, we investigated how one of the three spt15 mutants, spt15_Ala101Thr, was leading to enhanced isoprenoid pathway flux in S. cerevisiae. Metabolic flux analysis of the spt15_Ala101Thr mutant initially revealed a rerouting of the central carbon metabolism for the production of the precursor acetyl-CoA through activation of pyruvate-acetaldehyde-acetate cycle in the cytoplasm due to high flux in the reaction caused by pyruvate decarboxylase (PDC). This led to alternate routes of cytosolic NADPH generation, increased mitochondrial ATP production and phosphate demand in the mutant strain. Comparison of the transcriptomics of the spt15_Ala101Thr mutant cell with SPT15WT bearing cells shows upregulation of phosphate mobilization genes and pyruvate decarboxylase 6 (PDC6). Increasing the extracellular phosphate led to an increase in the growth rate and biomass but diverted flux away from the isoprenoid pathway. PDC6 is also shown to play a critical role in isoprenoid pathway flux under phosphate limitation conditions.
CONCLUSION
The study not only proposes a probable mechanism as to how a spt15_Ala101Thr mutant (a global TATA binding protein mutant) could increase flux towards the isoprenoid pathway, but also PDC as a new route of metabolic manipulation for increasing the isoprenoid flux in yeast.
Topics: Metabolic Engineering; Metabolic Networks and Pathways; Mutation; NADP; Phosphates; Pyruvate Decarboxylase; Saccharomyces cerevisiae; TATA-Box Binding Protein; Terpenes
PubMed: 30241525
DOI: 10.1186/s12934-018-1000-1 -
Bioengineered 2015Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae,...
Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved.
Topics: Acetic Acid; Gene Deletion; Genes, Fungal; Kinetics; Mannitol; Metabolic Engineering; Pyruvate Decarboxylase; Pyruvic Acid; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 26588105
DOI: 10.1080/21655979.2015.1112472 -
Engineering in Life Sciences Oct 2019Dicarboxylic acids are important bio-based building blocks, and is postulated to be an advantageous host for their fermentative production. Here, we engineered a...
Dicarboxylic acids are important bio-based building blocks, and is postulated to be an advantageous host for their fermentative production. Here, we engineered a pyruvate decarboxylase-negative strain for succinic acid production to exploit its promising properties, that is, lack of ethanol production and accumulation of the precursor pyruvate. The metabolic engineering steps included genomic integration of a biosynthesis pathway based on the reductive branch of the tricarboxylic acid cycle and a dicarboxylic acid transporter. Further modifications were the combined deletion of and and multi-copy integration of the native gene, encoding a pyruvate carboxylase required to drain pyruvate into the synthesis pathway. The effect of increased redox cofactor supply was tested by modulating oxygen limitation and supplementing formate. The physiologic analysis of the differently engineered strains focused on elucidating metabolic bottlenecks. The data not only highlight the importance of a balanced activity of pathway enzymes and selective export systems but also shows the importance to find an optimal trade-off between redox cofactor supply and energy availability in the form of ATP.
PubMed: 32624964
DOI: 10.1002/elsc.201900080 -
BMC Plant Biology Jun 2023Flooding is among the most severe abiotic stresses in plant growth and development. The mechanism of submergence tolerance of cotton in response to submergence stress is...
BACKGROUND
Flooding is among the most severe abiotic stresses in plant growth and development. The mechanism of submergence tolerance of cotton in response to submergence stress is unknown.
RESULTS
The transcriptome results showed that a total of 6,893 differentially expressed genes (DEGs) were discovered under submergence stress. Gene Ontology (GO) enrichment analysis showed that DEGs were involved in various stress or stimulus responses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that DEGs related to plant hormone signal transduction, starch and sucrose metabolism, glycolysis and the biosynthesis of secondary metabolites were regulated by submergence stress. Eight DEGs related to ethylene signaling and 3 ethylene synthesis genes were identified in the hormone signal transduction. For respiratory metabolism, alcohol dehydrogenase (ADH, GH_A02G0728) and pyruvate decarboxylase (PDC, GH_D09G1778) were significantly upregulated but 6-phosphofructokinase (PFK, GH_D05G0280), phosphoglycerate kinase (PGK, GH_A01G0945 and GH_D01G0967) and sucrose synthase genes (SUS, GH_A06G0873 and GH_D06G0851) were significantly downregulated in the submergence treatment. Terpene biosynthetic pathway-related genes in the secondary metabolites were regulated in submergence stress.
CONCLUSIONS
Regulation of terpene biosynthesis by respiratory metabolism may play a role in enhancing the tolerance of cotton to submergence under flooding. Our findings showed that the mevalonate pathway, which occurs in the cytoplasm of the terpenoid backbone biosynthesis pathway (ko00900), may be the main response to submergence stress.
Topics: Gene Expression Profiling; Transcriptome; Carbohydrate Metabolism; Stress, Physiological; Ethylenes; Gene Expression Regulation, Plant
PubMed: 37344795
DOI: 10.1186/s12870-023-04334-4 -
Applied Microbiology and Biotechnology Jul 2017The obligatory aerobic acetic acid bacterium Gluconobacter oxydans incompletely oxidizes carbon sources regio- and stereoselectively in the periplasm and therefore is...
The obligatory aerobic acetic acid bacterium Gluconobacter oxydans incompletely oxidizes carbon sources regio- and stereoselectively in the periplasm and therefore is used industrially for oxidative biotransformations, e. g., in vitamin C production. However, it has a very low biomass yield as the oxidized products largely remain in the medium and cannot be used for anabolism. Cytoplasmic carbon metabolism occurs via the pentose phosphate pathway and the Entner-Doudoroff pathway, whereas glycolysis and the tricarboxylic acid cycle are incomplete. Acetate is formed as an end product via pyruvate decarboxylase and acetaldehyde dehydrogenase. In order to increase the biomass yield from glucose, we sequentially replaced (i) gdhS encoding the cytoplasmic NADP-dependent glucose dehydrogenase by the Acetobacter pasteurianus sdhCDABE genes for succinate dehydrogenase and the flavinylation factor SdhE (strain IK001), (ii) pdc encoding pyruvate decarboxylase by a second ndh gene encoding a type II NADH dehydrogenase (strain IK002.1), and (iii) gdhM encoding the membrane-bound PQQ-dependent glucose dehydrogenase by sucCD from Gluconacetobacter diazotrophicus encoding succinyl-CoA synthetase (strain IK003.1). Analysis of the strains under controlled cultivation conditions in bioreactors revealed for IK003.1 that neither gluconate nor 2-ketogluconate was formed, but some 5-ketogluconate. Acetate formation was eliminated, and comparable amounts of pyruvate were formed instead. CO formation by IK003.1 was more than doubled compared to the reference strain. Growth of IK003.1 was retarded, but the biomass yield of this strain was raised by 60%. IK003.1 serves as suitable host for oxidative biotransformations and for further metabolic engineering.
Topics: Acetobacter; Acyl Coenzyme A; Bacterial Proteins; Biomass; Bioreactors; Citric Acid Cycle; Gluconobacter oxydans; Glucose; Glucose 1-Dehydrogenase; Glycolysis; Metabolic Engineering; Oxidation-Reduction; Pyruvate Decarboxylase; Succinate Dehydrogenase; Sugar Alcohol Dehydrogenases
PubMed: 28484812
DOI: 10.1007/s00253-017-8308-3 -
Biology Jan 2022Waterlogging is one of the serious abiotic stresses that inhibits crop growth and reduces productivity. Therefore, investigating efficient waterlogging mitigation...
Waterlogging is one of the serious abiotic stresses that inhibits crop growth and reduces productivity. Therefore, investigating efficient waterlogging mitigation measures has both theoretical and practical significance. The objectives of the present research were to examine the efficiency of melatonin and KNO seed soaking and foliar application on alleviating the waterlogging inhibited growth performance of maize seedlings. In this study, 100 µM melatonin and different levels (0.25, 0.50 and 0.75 g) of potassium nitrate (KNO) were used in seed soaking and foliar applications. For foliar application, treatments were applied at the 7th leaf stage one week after the imposition of waterlogging stress. The results showed that melatonin with KNO significantly improved the plant growth and biochemical parameters of maize seedlings under waterlogging stress conditions. However, the application of melatonin with KNO treatments increased plant growth characteristics, chlorophyll content, and the net photosynthetic rate at a variable rate under waterlogging stress. Furthermore, melatonin with KNO treatments significantly reduced the accumulation of hydrogen peroxide (HO) and malondialdehyde (MDA), and it decreased the activity of pyruvate decarboxylase and alcohol dehydrogenase, while increasing enzymatic activities and soluble protein content of maize seedlings under waterlogging stress conditions. Overall, our results indicated that seed soaking with 100 µM melatonin and 0.50 g KNO was the most effective treatment that significantly improved the plant growth characteristics, chlorophyll content, photosynthetic rate, and enzymatic activity of maize seedling under waterlogging conditions.
PubMed: 35053096
DOI: 10.3390/biology11010099 -
Journal of Biochemistry Dec 2015Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed...
Molecular and biochemical characterization of bifunctional pyruvate decarboxylases and pyruvate ferredoxin oxidoreductases from Thermotoga maritima and Thermotoga hypogea.
Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea. Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles.
Topics: Acetaldehyde; Bacterial Proteins; Enzyme Stability; Ethanol; Hydrogen-Ion Concentration; Kinetics; Multifunctional Enzymes; Pyruvate Decarboxylase; Pyruvate Synthase; Pyruvic Acid; Temperature; Thermotoga maritima
PubMed: 26032540
DOI: 10.1093/jb/mvv058 -
BMC Biotechnology May 2016The twin problem of shortage in fossil fuel and increase in environmental pollution can be partly addressed by blending of ethanol with transport fuel. Increasing the...
BACKGROUND
The twin problem of shortage in fossil fuel and increase in environmental pollution can be partly addressed by blending of ethanol with transport fuel. Increasing the ethanol production for this purpose without affecting the food security of the countries would require the use of cellulosic plant materials as substrate. Clostridium thermocellum is an anaerobic thermophilic bacterium with cellulolytic property and the ability to produce ethanol. But its application as biocatalyst for ethanol production is limited because pyruvate ferredoxin oxidoreductase, which diverts pyruvate to ethanol production pathway, has low affinity to the substrate. Therefore, the present study was undertaken to genetically modify C. thermocellum for enhancing its ethanol production capacity by transferring pyruvate carboxylase (pdc) and alcohol dehydrogenase (adh) genes of the homoethanol pathway from Zymomonas mobilis.
RESULTS
The pdc and adh genes from Z. mobilis were cloned in pNW33N, and transformed to Clostridium thermocellum DSM 1313 by electroporation to generate recombinant CTH-pdc, CTH-adh and CTH-pdc-adh strains that carried heterologous pdc, adh, and both genes, respectively. The plasmids were stably maintained in the recombinant strains. Though both pdc and adh were functional in C. thermocellum, the presence of adh severely limited the growth of the recombinant strains, irrespective of the presence or absence of the pdc gene. The recombinant CTH-pdc strain showed two-fold increase in pyruvate carboxylase activity and ethanol production when compared with the wild type strain.
CONCLUSIONS
Pyruvate decarboxylase gene of the homoethanol pathway from Z mobilis was functional in recombinant C. thermocellum strain and enhanced its ability to produced ethanol. Strain improvement and bioprocess optimizations may further increase the ethanol production from this recombinant strain.
Topics: Alcohol Dehydrogenase; Bioreactors; Clostridium thermocellum; Ethanol; Genetic Enhancement; Metabolic Engineering; Pyruvate Carboxylase; Recombinant Proteins; Species Specificity; Zymomonas
PubMed: 27213504
DOI: 10.1186/s12896-016-0260-2 -
Applied Microbiology and Biotechnology Aug 2023Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies...
Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies in cell growth and D-lactate production at high substrate concentrations. Complex nutrients or high cell density were thus required to support growth and D-lactate production with a potential to increase medium and process cost of industrial-scale D-lactate production. As an alternative microbial biocatalyst, a Crabtree-negative and thermotolerant yeast Kluyveromyces marxianus was engineered in this study to produce high titer and yield of D-lactate at a lower pH without growth defects. Only pyruvate decarboxylase 1 (PDC1) gene was replaced by a codon-optimized bacterial D-lactate dehydrogenase (ldhA). Ethanol, glycerol, or acetic acid was not produced by the resulting strain, KMΔpdc1::ldhA. Aeration rate at 1.5 vvm and culture pH 5.0 at 30 °C provided the highest D-lactate titer of 42.97 ± 0.48 g/L from glucose. Yield and productivity of D-lactate, and glucose-consumption rate were 0.85 ± 0.01 g/g, 0.90 ± 0.01 g/(L·h), and 1.06 ± 0.00 g/(L·h), respectively. Surprisingly, D-lactate titer, productivity, and glucose-consumption rate of 52.29 ± 0.68 g/L, 1.38 ± 0.05 g/(L·h), and 1.22 ± 0.00 g/(L·h), respectively, were higher at 42 °C compared to 30 °C. Sugarcane molasses, a low-value carbon, led to the highest D-lactate titer and yield of 66.26 ± 0.81 g/L and 0.91 ± 0.01 g/g, respectively, in a medium without additional nutrients. This study is a pioneer work of engineering K. marxianus to produce D-lactate at the yield approaching theoretical maximum using simple batch process. Our results support the potential of an engineered K. marxianus for D-lactate production on an industrial scale. KEY POINTS: • K. marxianus was engineered by deleting PDC1 and expressing codon-optimized D-ldhA. • The strain allowed high D-lactate titer and yield under pH ranging from 3.5 to 5.0. • The strain produced 66 g/L D-lactate at 30 °C from molasses without any additional nutrients.
Topics: Lactic Acid; Saccharomyces cerevisiae; Kluyveromyces; L-Lactate Dehydrogenase; Glucose; Pyruvate Decarboxylase; Hydrogen-Ion Concentration; Fermentation
PubMed: 37405435
DOI: 10.1007/s00253-023-12658-2