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Life Sciences in Space Research Feb 2016Establishing plants in space, Moon or Mars requires adaptation to altered conditions, including reduced pressure and composition of atmospheres. To determine the oxygen...
Establishing plants in space, Moon or Mars requires adaptation to altered conditions, including reduced pressure and composition of atmospheres. To determine the oxygen requirements for seed germination, we imbibed Brassica rapa seeds under varying oxygen concentrations and profiled the transcription patterns of genes related to early metabolism such as starch degradation, glycolysis, and fermentation. We also analyzed the activity of lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH), and measured starch degradation. Partial oxygen pressure (pO2) greater than 10% resulted in normal germination (i.e., protrusion of radicle about 18 hours after imbibition) but lower pO2 delayed and reduced germination. Imbibition in an oxygen-free atmosphere for three days resulted in no germination but subsequent transfer to air initiated germination in 75% of the seeds and the root growth rate was transiently greater than in roots germinated under ambient pO2. In hypoxic seeds soluble sugars degraded faster but the content of starch after 24 h was higher than at ambient oxygen. Transcription of genes related to starch degradation, α-amylase (AMY) and Sucrose Synthase (SUS), was higher under ambient O2 than under hypoxia. Glycolysis and fermentation pathway-related genes, glucose phosphate isomerase (GPI), 6-phosphofructokinase (PFK), fructose 1,6-bisphosphate aldolase (ALD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate decarboxylase (PDC), LDH, and ADH, were induced by low pO2. The activity of LDH and ADH was the highest in anoxic seeds. Germination under low O2 conditions initiated ethanolic fermentation. Therefore, sufficient oxygen availability is important for germination before photosynthesis provides necessary oxygen and the determination of an oxygen carrying capacity is important for uniform growth in space conditions.
Topics: Alcohol Dehydrogenase; Brassica; Gene Expression Regulation, Plant; Germination; Oxygen; Oxygen Consumption; Seeds; Starch; alpha-Amylases
PubMed: 26948011
DOI: 10.1016/j.lssr.2016.01.002 -
Frontiers in Oncology 2018Glycine decarboxylase (GLDC) gene is frequently upregulated in various types of cancer including lung, prostate and brain. It catabolizes glycine to yield...
Glycine decarboxylase (GLDC) gene is frequently upregulated in various types of cancer including lung, prostate and brain. It catabolizes glycine to yield 5,10-methylenetetrahydrofolate, an important substrate in one-carbon metabolism for nucleotide synthesis. In this study, we used exon splicing modulating steric hindrance antisense oligonucleotide (shAON) to suppress GLDC expression and investigated its effect on pyruvate metabolism hyperpolarized carbon-13 magnetic resonance spectroscopy (MRS). The MRS technique allows us to study metabolic flux in tumor tissues with/without GLDC-shAON intervention. Here, we show that GLDC-shAON treatment is able to suppress lung cancer cell growth and tumorigenesis, both and . The carbon-13 MRS results indicated that the conversion of pyruvate into lactate in GLDC-shAON-treated tumor tissues was significantly reduced, when compared with the control groups. This observation corroborated with the reduced activity of lactate dehydrogenase and pyruvate dehydrogenase in GLDC-shAON-treated lung cancer cells and tumor tissues. Glycolysis stress test showed that extracellular acidification rate was significantly suppressed after GLDC-shAON treatment. Besides lung cancer, the antitumor effect of GLDC-shAON was also observed in brain, liver, cervical, and prostate cancer cell lines. Furthermore, it enhanced the treatment efficacy of cisplatin in lung cancer cells. Taken together, our findings illustrate that pyruvate metabolism decreases upon GLDC inhibition, thereby starving cancer cells from critical metabolic fuels.
PubMed: 29911072
DOI: 10.3389/fonc.2018.00196 -
Biotechnology For Biofuels and... Mar 2023Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy....
BACKGROUND
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.
RESULTS
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 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 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 sugars and resulted in a final titer of 36.0 g/L 3-HP.
CONCLUSIONS
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.
PubMed: 36991437
DOI: 10.1186/s13068-023-02288-1 -
Plant Physiology Mar 2021Sugar supply is a key component of hypoxia tolerance and acclimation in plants. However, a striking gap remains in our understanding of mechanisms governing sugar...
Sugar supply is a key component of hypoxia tolerance and acclimation in plants. However, a striking gap remains in our understanding of mechanisms governing sugar impacts on low-oxygen responses. Here, we used a maize (Zea mays) root-tip system for precise control of sugar and oxygen levels. We compared responses to oxygen (21 and 0.2%) in the presence of abundant versus limited glucose supplies (2.0 and 0.2%). Low-oxygen reconfigured the transcriptome with glucose deprivation enhancing the speed and magnitude of gene induction for core anaerobic proteins (ANPs). Sugar supply also altered profiles of hypoxia-responsive genes carrying G4 motifs (sources of regulatory quadruplex structures), revealing a fast, sugar-independent class followed more slowly by feast-or-famine-regulated G4 genes. Metabolite analysis showed that endogenous sugar levels were maintained by exogenous glucose under aerobic conditions and demonstrated a prominent capacity for sucrose re-synthesis that was undetectable under hypoxia. Glucose abundance had distinctive impacts on co-expression networks associated with ANPs, altering network partners and aiding persistence of interacting networks under prolonged hypoxia. Among the ANP networks, two highly interconnected clusters of genes formed around Pyruvate decarboxylase 3 and Glyceraldehyde-3-phosphate dehydrogenase 4. Genes in these clusters shared a small set of cis-regulatory elements, two of which typified glucose induction. Collective results demonstrate specific, previously unrecognized roles of sugars in low-oxygen responses, extending from accelerated onset of initial adaptive phases by starvation stress to maintenance and modulation of co-expression relationships by carbohydrate availability.
Topics: Anaerobiosis; Glucose; Glyceraldehyde-3-Phosphate Dehydrogenases; Oxygen; Plant Proteins; Plant Roots; Pyruvate Decarboxylase; Stress, Physiological; Sugars; Transcriptome; Zea mays
PubMed: 33721892
DOI: 10.1093/plphys/kiaa029 -
Life (Basel, Switzerland) Apr 2023Mulberry (), a widely distributed economic plant, can withstand long-term flooding stress. However, the regulatory gene network underlying this tolerance is unknown. In...
Mulberry (), a widely distributed economic plant, can withstand long-term flooding stress. However, the regulatory gene network underlying this tolerance is unknown. In the present study, mulberry plants were subjected to submergence stress. Subsequently, mulberry leaves were collected to perform quantitative reverse-transcription PCR (qRT-PCR) and transcriptome analysis. Genes encoding ascorbate peroxidase and glutathione S-transferase were significantly upregulated after submergence stress, indicating that they could protect the mulberry plant from flood damage by mediating ROS homeostasis. Genes that regulate starch and sucrose metabolism; genes encoding pyruvate kinase, alcohol dehydrogenase, and pyruvate decarboxylase (enzymes involved in glycolysis and ethanol fermentation); and genes encoding malate dehydrogenase and ATPase (enzymes involved in the TCA cycle) were also obviously upregulated. Hence, these genes likely played a key role in mitigating energy shortage during flooding stress. In addition, genes associated with ethylene, cytokinin, abscisic acid, and MAPK signaling; genes involved in phenylpropanoid biosynthesis; and transcription factor genes also showed upregulation under flooding stress in mulberry plants. These results provide further insights into the adaptation mechanisms and genetics of submergence tolerance in mulberry plants and could aid in the molecular breeding of these plants.
PubMed: 37240733
DOI: 10.3390/life13051087 -
ACS Synthetic Biology Dec 2014Ethanol is an important biofuel. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) increases ethanol production...
Ethanol is an important biofuel. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) increases ethanol production in Escherichia coli. A fusion of PDC and ADH was generated and expressed in E. coli. The fusion enzyme was demonstrated to possess both activities. AdhB activity was significantly lower when fused to PDC than when the two enzymes were expressed separately. However, cells expressing the fusion protein generated ethanol more rapidly and to higher levels than cells coexpressing Pdc and AdhB, suggesting a specific rate enhancement due to the fusion of the two enzymes.
Topics: Alcohol Dehydrogenase; Escherichia coli; Ethanol; Models, Molecular; Pyruvate Decarboxylase; Recombinant Fusion Proteins
PubMed: 25524103
DOI: 10.1021/sb500020g -
Scientific Reports Aug 2019In this work, we describe the construction of a synthetic metabolic pathway enabling direct biosynthesis of 1,3-propanediol (PDO) from glucose via the Krebs cycle...
In this work, we describe the construction of a synthetic metabolic pathway enabling direct biosynthesis of 1,3-propanediol (PDO) from glucose via the Krebs cycle intermediate malate. This non-natural pathway extends a previously published synthetic pathway for the synthesis of (L)-2,4-dihydroxybutyrate (L-DHB) from malate by three additional reaction steps catalyzed respectively, by a DHB dehydrogenase, a 2-keto-4-hydroxybutyrate (OHB) dehydrogenase and a PDO oxidoreductase. Screening and structure-guided protein engineering provided a (L)-DHB dehydrogenase from the membrane-associated (L)-lactate dehydrogenase of E. coli and OHB decarboxylase variants derived from the branched-chain keto-acid decarboxylase encoded by kdcA from Lactococcus lactis or pyruvate decarboxylase from Zymomonas mobilis. The simultaneous overexpression of the genes encoding these enzymes together with the endogenous ydhD-encoded aldehyde reductase enabled PDO biosynthesis from (L)-DHB. While the simultaneous expression of the six enzymatic activities in a single engineered E. coli strain resulted in a low production of 0.1 mM PDO from 110 mM glucose, a 40-fold increased PDO titer was obtained by co-cultivation of an E. coli strain expressing the malate-DHB pathway with another strain harboring the DHB-to-PDO pathway.
Topics: Biosynthetic Pathways; Citric Acid Cycle; Escherichia coli; Glucose; Industrial Microbiology; Lactococcus lactis; Metabolic Engineering; Propylene Glycols; Pyruvate Decarboxylase; Zymomonas
PubMed: 31399628
DOI: 10.1038/s41598-019-48091-7 -
Journal of Experimental Botany Mar 2019Ethanol fermentation is considered as one of the main metabolic adaptations to ensure energy production in higher plants under anaerobic conditions. Following this...
Ethanol fermentation is considered as one of the main metabolic adaptations to ensure energy production in higher plants under anaerobic conditions. Following this pathway, pyruvate is decarboxylated and reduced to ethanol with the concomitant oxidation of NADH to NAD+. Despite its acknowledgement as an essential metabolic strategy, the conservation of this pathway and its regulation throughout plant evolution have not been assessed so far. To address this question, we compared ethanol fermentation in species representing subsequent steps in plant evolution and related it to the structural features and transcriptional regulation of the two enzymes involved: pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). We observed that, despite the conserved ability to produce ethanol upon hypoxia in distant phyla, transcriptional regulation of the enzymes involved is not conserved in ancient plant lineages, whose ADH homologues do not share structural features distinctive for acetaldehyde/ethanol-processing enzymes. Moreover, Arabidopsis mutants devoid of ADH expression exhibited enhanced PDC activity and retained substantial ethanol production under hypoxic conditions. Therefore, we concluded that, whereas ethanol production is a highly conserved adaptation to low oxygen, its catalysis and regulation in land plants probably involve components that will be identified in the future.
Topics: Alcohol Dehydrogenase; Biological Evolution; Embryophyta; Ethanol; Fermentation; Pyruvate Decarboxylase
PubMed: 30861072
DOI: 10.1093/jxb/erz052 -
Engineering in Life Sciences Jul 2019Pyruvate decarboxylase (PDC) is responsible for the decarboxylation of pyruvate, producing acetaldehyde and carbon dioxide and is of high interest for industrial...
Pyruvate decarboxylase (PDC) is responsible for the decarboxylation of pyruvate, producing acetaldehyde and carbon dioxide and is of high interest for industrial applications. PDC is a very powerful tool in the enzymatic synthesis of chiral amines by combining it with transaminases when alanine is used as amine donor. However, one of the main drawback that hampers its use in biocatalysis is its production and the downstream processing on scale. In this paper, a production process of PDC from has been developed. The enzyme has been cloned and overexpressed in . It is presented, for the first time, the evaluation of the production of recombinant PDC in a bench-scale bioreactor, applying a substrate-limiting fed-batch strategy which led to a volumetric productivity and a final PDC specific activity of 6942 U Lh and 3677 U gDCW (dry cell weight). Finally, PDC was purified in fast protein liquid chromatography equipment by ion exchange chromatography. The developed purification process resulted in 100% purification yield and a purification factor of 3.8.
PubMed: 32625027
DOI: 10.1002/elsc.201900010 -
Preparative Biochemistry & Biotechnology 2022has good reproductive ability in both haploid and diploid forms, a pyruvate decarboxylase plays an important role in cell metabolism. In this study, 1 and 5 double...
has good reproductive ability in both haploid and diploid forms, a pyruvate decarboxylase plays an important role in cell metabolism. In this study, 1 and 5 double knockout strains of H14-02 (a type) and H5-02 (α type) were obtained by the Cre/loxP technique. The effects of the deletion of 1 and 5 on the metabolites of the two haploid strains were consistent. In H14-02, the ethanol conversion decreased by 30.19%, the conversion of glycerol increased by 40.005%, the concentration of acetic acid decreased by 43.54%, the concentration of acetoin increased by 12.79 times, and the activity of pyruvate decarboxylase decreased by 40.91% compared to those in the original H14 strain. The original haploid strain H14 produced a small amount of acetoin but produced very little 2,3-butanediol. However, H14-02 produced 1.420 ± 0.063 g/L 2,3-BD. This study not only provides strain selection for obtaining haploid strains with a high yield of 2,3-BD but also lays a foundation for haploid to be used as a new tool for genetic research and breeding programs.
Topics: Acetoin; Butylene Glycols; Carboxy-Lyases; Ethanol; Gene Deletion; Gene Expression Regulation, Fungal; Gene Knockout Techniques; Glycerol; Haploidy; Pyruvate Decarboxylase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 33881948
DOI: 10.1080/10826068.2021.1910958