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Applied Microbiology and Biotechnology Apr 2023β-alanine has been used in food and pharmaceutical industries. Although Escherichia coli Nissle 1917 (EcN) is generally considered safe and engineered as living...
β-alanine has been used in food and pharmaceutical industries. Although Escherichia coli Nissle 1917 (EcN) is generally considered safe and engineered as living therapeutics, engineering EcN for producing industrial metabolites has rarely been explored. Here, by protein and metabolic engineering, EcN was engineered for producing β-alanine from glucose. First, an aspartate-α-decarboxylase variant ADC with improved activity was identified and over-expressed in EcN, promoting the titer of β-alanine from an undetectable level to 0.46 g/L. Second, directing the metabolic flux towards L-aspartate increased the titer of β-alanine to 0.92 g/L. Third, the yield of β-alanine was elevated to 1.19 g/L by blocking conversion of phosphoenolpyruvate to pyruvate, and further increased to 2.14 g/L through optimizing culture medium. Finally, the engineered EcN produced 11.9 g/L β-alanine in fed-batch fermentation. Our work not only shows the potential of EcN as a valuable industrial platform, but also facilitates production of β-alanine via fermentation. KEY POINTS: • Escherichia coli Nissle 1917 (EcN) was engineered as a β-alanine producing cell factory • Identification of a decarboxylase variant ADC with improved activity • Directing the metabolic flux to L-ASP and expressing ADC elevated the titer of β-alanine to 11.9 g/L.
Topics: Metabolic Engineering; Escherichia coli; Probiotics; beta-Alanine; Fermentation
PubMed: 36929190
DOI: 10.1007/s00253-023-12477-5 -
International Journal of Molecular... Feb 2023The major oxidized product of cholesterol, 7-Ketocholesterol (7KCh), causes cellular oxidative damage. In the present study, we investigated the physiological responses...
The major oxidized product of cholesterol, 7-Ketocholesterol (7KCh), causes cellular oxidative damage. In the present study, we investigated the physiological responses of cardiomyocytes to 7KCh. A 7KCh treatment inhibited the growth of cardiac cells and their mitochondrial oxygen consumption. It was accompanied by a compensatory increase in mitochondrial mass and adaptive metabolic remodeling. The application of [U-C] glucose labeling revealed an increased production of malonyl-CoA but a decreased formation of hydroxymethylglutaryl-coenzyme A (HMG-CoA) in the 7KCh-treated cells. The flux of the tricarboxylic acid (TCA) cycle decreased, while that of anaplerotic reaction increased, suggesting a net conversion of pyruvate to malonyl-CoA. The accumulation of malonyl-CoA inhibited the carnitine palmitoyltransferase-1 (CPT-1) activity, probably accounting for the 7-KCh-induced suppression of β-oxidation. We further examined the physiological roles of malonyl-CoA accumulation. Treatment with the inhibitor of malonyl-CoA decarboxylase, which increased the intracellular malonyl-CoA level, mitigated the growth inhibitory effect of 7KCh, whereas the treatment with the inhibitor of acetyl-CoA carboxylase, which reduced malonyl-CoA content, aggravated such a growth inhibitory effect. Knockout of malonyl-CoA decarboxylase gene () alleviated the growth inhibitory effect of 7KCh. It was accompanied by improvement of the mitochondrial functions. These findings suggest that the formation of malonyl-CoA may represent a compensatory cytoprotective mechanism to sustain the growth of 7KCh-treated cells.
Topics: Humans; Malonyl Coenzyme A; Carnitine O-Palmitoyltransferase; Heart; Growth Disorders
PubMed: 36901848
DOI: 10.3390/ijms24054418 -
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 -
Computational and Structural... 2023Short-chain fatty acids (SCFAs) exhibit anticancer activity in cellular and animal models of colon cancer. Acetate, propionate, and butyrate are the three major SCFAs...
Short-chain fatty acids (SCFAs) exhibit anticancer activity in cellular and animal models of colon cancer. Acetate, propionate, and butyrate are the three major SCFAs produced from dietary fiber by gut microbiota fermentation and have beneficial effects on human health. Most previous studies on the antitumor mechanisms of SCFAs have focused on specific metabolites or genes involved in antitumor pathways, such as reactive oxygen species (ROS) biosynthesis. In this study, we performed a systematic and unbiased analysis of the effects of acetate, propionate, and butyrate on ROS levels and metabolic and transcriptomic signatures at physiological concentrations in human colorectal adenocarcinoma cells. We observed significantly elevated levels of ROS in the treated cells. Furthermore, significantly regulated signatures were involved in overlapping pathways at metabolic and transcriptomic levels, including ROS response and metabolism, fatty acid transport and metabolism, glucose response and metabolism, mitochondrial transport and respiratory chain complex, one-carbon metabolism, amino acid transport and metabolism, and glutaminolysis, which are directly or indirectly linked to ROS production. Additionally, metabolic and transcriptomic regulation occurred in a SCFAs types-dependent manner, with an increasing degree from acetate to propionate and then to butyrate. This study provides a comprehensive analysis of how SCFAs induce ROS production and modulate metabolic and transcriptomic levels in colon cancer cells, which is vital for understanding the mechanisms of the effects of SCFAs on antitumor activity in colon cancer.
PubMed: 36874158
DOI: 10.1016/j.csbj.2023.02.022 -
Biotechnology Journal Apr 2023Engineered Saccharomyces cerevisiae expressing a lactic acid dehydrogenase can metabolize pyruvate into lactic acid. However, three pyruvate decarboxylase (PDC) isozymes...
Engineered Saccharomyces cerevisiae expressing a lactic acid dehydrogenase can metabolize pyruvate into lactic acid. However, three pyruvate decarboxylase (PDC) isozymes drive most carbon flux toward ethanol rather than lactic acid. Deletion of endogenous PDCs will eliminate ethanol production, but the resulting strain suffers from C auxotrophy and struggles to complete a fermentation. Engineered yeast assimilating xylose or cellobiose produce lactic acid rather than ethanol as a major product without the deletion of any PDC genes. We report here that sugar flux, but not sensing, contributes to the partition of flux at the pyruvate branch point in S. cerevisiae expressing the Rhizopus oryzae lactic acid dehydrogenase (LdhA). While the membrane glucose sensors Snf3 and Rgt2 did not play any direct role in the option of predominant product, the sugar assimilation rate was strongly correlated to the partition of flux at pyruvate: fast sugar assimilation favors ethanol production while slow sugar assimilation favors lactic acid. Applying this knowledge, we created an engineered yeast capable of simultaneously converting glucose and xylose into lactic acid, increasing lactic acid production to approximately 17 g L from the 12 g L observed during sequential consumption of sugars. This work elucidates the carbon source-dependent effects on product selection in engineered yeast.
Topics: Saccharomyces cerevisiae; Glucose; Lactic Acid; Xylose; Pyruvic Acid; Ethanol; Fermentation; Oxidoreductases
PubMed: 36723451
DOI: 10.1002/biot.202200535 -
Scientific Reports Jan 2023Open burning of agricultural residues causes numerous complications including particulate matter pollution in the air, soil degradation, global warming and many more....
Open burning of agricultural residues causes numerous complications including particulate matter pollution in the air, soil degradation, global warming and many more. Since they possess bio-conversion potential, agro-industrial residues including sugarcane bagasse (SCB), rice straw (RS), corncob (CC) and sweet sorghum bagasse (SSB) were chosen for the study. Yeast strains, Candida tropicalis, C. shehatae, Saccharomyces cerevisiae, and Kluyveromyces marxianus var. marxianus were compared for their production potential of bioethanol and phenylacetylcarbinol (PAC), an intermediate in the manufacture of crucial pharmaceuticals, namely, ephedrine, and pseudoephedrine. Among the substrates and yeasts evaluated, RS cultivated with C. tropicalis produced significantly (p ≤ 0.05) higher ethanol concentration at 15.3 g L after 24 h cultivation. The product per substrate yield (Y) was 0.38 g g with the volumetric productivity (Q) of 0.64 g L h and fermentation efficiency of 73.6% based on a theoretical yield of 0.51 g ethanol/g glucose. C. tropicalis grown in RS medium produced 0.303 U mL pyruvate decarboxylase (PDC), a key enzyme that catalyzes the production of PAC, with a specific activity of 0.400 U mg protein after 24 h cultivation. This present study also compared the whole cells biomass of C. tropicalis with its partially purified PDC preparation for PAC biotransformation. The whole cells C. tropicalis PDC at 1.29 U mL produced an overall concentration of 62.3 mM PAC, which was 68.4% higher when compared to partially purified enzyme preparation. The results suggest that the valorization of lignocellulosic residues into bioethanol and PAC will not only aid in mitigating the environmental challenge posed by their surroundings but also has the potential to improve the bioeconomy.
Topics: Cellulose; Oryza; Sorghum; Saccharum; Fermentation; Saccharomyces cerevisiae; Candida tropicalis; Ethanol
PubMed: 36639688
DOI: 10.1038/s41598-023-27451-4 -
Journal of Food Science and Technology Jan 2023Development of fermented flavour during storage reduces acceptability of Shughri pear. Therefore, the current study was designed to investigate the combined effect of...
Development of fermented flavour during storage reduces acceptability of Shughri pear. Therefore, the current study was designed to investigate the combined effect of 1-Methylcyclopropene (1-MCP) and hypobaric treatment on stability of Shughri pear during 120 days of storage. Fruit were treated individually or combinedly with 25, 50, and 75 kilo pascal hypobaric treatments for 4 h and 1-MCP (0.3 μLL and 0.6 μLL) for 24 h, whereas control received no treatment. The pears were stored for 120 days at (0 ± 1 °C, 85 ± 5% RH), and were evaluated after every 30 days. After cold storage, pears were shifted to simulated retail conditions (20 ± 3 °C, 65 ± 5% RH). The combination of 25 kPa + 0.6 μLL 1-MCP significantly (P ≤ 0.05) delayed fruit ripening, reduced Alcohol dehydrogenase (ADH), and Pyruvate decarboxylase (PDC) activities, maintained the quality, and led to higher consumers' acceptability of the pear followed by 50 kPa + 0.6 μLL and 25 kPa + 0.3 μLL. The control fruit were marketable for a week after storage with relatively less acceptability due to fermented flavour compared to treated fruit, marketable for more than two weeks. Among all the treatments, the synergy of 1-MCP and hypobaric treatment 25 kPa + 0.6 μLL 1-MCP improved the postharvest storage life and quality parameters, preventing development of fermented flavour in the pears. The experiment was conducted on pilot scale, for commercial application, the results of this study should be validated on large scale.
PubMed: 36618031
DOI: 10.1007/s13197-022-05605-y -
The Journal of Biological Chemistry Dec 2022Pyruvate has two major fates upon entry into mitochondria, the oxidative decarboxylation to acetyl-CoA via the pyruvate decarboxylase complex or the biotin-dependent...
Pyruvate has two major fates upon entry into mitochondria, the oxidative decarboxylation to acetyl-CoA via the pyruvate decarboxylase complex or the biotin-dependent carboxylation to oxaloacetate via pyruvate carboxylase (Pcx). Here, we have generated mice with a liver-specific KO of pyruvate carboxylase (Pcx) to understand the role of Pcx in hepatic mitochondrial metabolism under disparate physiological states. Pcx mice exhibited a deficit in hepatic gluconeogenesis and enhanced ketogenesis as expected but were able to maintain systemic euglycemia following a 24 h fast. Feeding a high-fat diet to Pcx mice resulted in animals that were resistant to glucose intolerance without affecting body weight. However, we found that Pcx mice fed a ketogenic diet for 1 week became severely hypoglycemic, demonstrating a requirement for hepatic Pcx for long-term glycemia under carbohydrate-limited diets. Additionally, we determined that loss of Pcx was associated with an induction in the abundance of lysine-acetylated proteins in Pcx mice regardless of physiologic state. Furthermore, liver acetyl-proteomics revealed a biased induction in mitochondrial lysine-acetylated proteins. These data show that Pcx is important for maintaining the proper balance of pyruvate metabolism between oxidative and anaplerotic pathways.
Topics: Animals; Mice; Diet, Ketogenic; Fasting; Gluconeogenesis; Liver; Lysine; Pyruvate Carboxylase; Pyruvic Acid
PubMed: 36441025
DOI: 10.1016/j.jbc.2022.102648 -
Frontiers in Plant Science 2022Quinoa ( Willd.), an Andean native crop, is increasingly popular around the world due to its high nutritional content and stress tolerance. The production and the...
Quinoa ( Willd.), an Andean native crop, is increasingly popular around the world due to its high nutritional content and stress tolerance. The production and the popularity of this strategic global food are greatly restricted by many limiting factors, such as seed pre-harvest sprouting, bitter saponin, . To solve these problems, the underlying mechanism of seed maturation in quinoa needs to be investigated. In this study, based on the investigation of morphological characteristics, a quantitative analysis of its global proteome was conducted using the combinational proteomics of tandem mass tag (TMT) labeling and parallel reaction monitoring (PRM). The proteome changes related to quinoa seed maturation conversion were monitored to aid its genetic improvement. Typical changes of morphological characteristics were discovered during seed maturation, including mean grain diameter, mean grain thickness, mean hundred-grain weight, palea, episperm color, . With TMT proteomics analysis, 581 differentially accumulated proteins (DAPs) were identified. Functional classification analysis and Gene Ontology enrichment analysis showed that most DAPs involved in photosynthesis were downregulated, indicating low levels of photosynthesis. DAPs that participated in glycolysis, such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate decarboxylase, and alcohol dehydrogenase, were upregulated to fulfill the increasing requirement of energy consumption during maturation conversion. The storage proteins, such as globulins, legumins, vicilins, and oleosin, were also increased significantly during maturation conversion. Protein-protein interaction analysis and function annotation revealed that the upregulation of oleosin, oil body-associated proteins, and acyl-coenzyme A oxidase 2 resulted in the accumulation of oil in quinoa seeds. The downregulation of β-amyrin 28-oxidase was observed, indicating the decreasing saponin content, during maturation, which makes the quinoa "sweet". By the PRM and qRT-PCR analysis, the expression patterns of most selected DAPs were consistent with the result of TMT proteomics. Our study enhanced the understanding of the maturation conversion in quinoa. This might be the first and most important step toward the genetic improvement of quinoa.
PubMed: 36426144
DOI: 10.3389/fpls.2022.975073 -
Frontiers in Plant Science 2022This research focused on cadmium (Cd), which negatively affects plant growth and auxin hemostasis. In plants, many processes are indirectly controlled through the...
Indole pyruvate decarboxylase gene regulates the auxin synthesis pathway in rice by interacting with the indole-3-acetic acid-amido synthetase gene, promoting root hair development under cadmium stress.
This research focused on cadmium (Cd), which negatively affects plant growth and auxin hemostasis. In plants, many processes are indirectly controlled through the expression of certain genes due to the secretion of bacterial auxin, as indole-3-acetic acid (IAA) acts as a reciprocal signaling molecule in plant-microbe interaction. The aim of current studies was to investigate responsible genes in rice for plant-microbe interaction and lateral root development due to the involvement of several metabolic pathways. Studies revealed that interacts with endogenous IAA in a homeostasis manner without directly providing IAA. In rice, indole-3-pyruvate decarboxylase () transgenic lines showed a 40% increase in lateral roots. Auxin levels and YUCCA (auxin biosynthesis gene) expression were monitored in mutant lines inoculated with exposed to Cd. The results showed an increase in root hairs (RHs) and lateral root density, changes in auxin levels, and expression of the YUCCA gene. normalizes the oxidative stress caused by Cd due to the accumulation of and HO in mutant lines. Furthermore, the inoculation increases DR5:GUS expression, indicating that bacterial species have a positive role in auxin regulation. Thus, the current study suggests that and transgenic lines increase the RH development in rice by interacting with IAA synthetase genes in the host plant, alleviating Cd toxicity and enhancing plant defense mechanisms.
PubMed: 36340357
DOI: 10.3389/fpls.2022.1023723