Pyruvate Decarboxylase Activity Research Articles

Pyruvate decarboxylase in minimally processed young palm cladode

The palm is a cactaceous of great global importance, being the young cladodes minimally processed a viable alternative consumption for cooking. Among the studied palm variables, enzymes play a major role in the post-harvest quality of these species, generating oxidation and influencing the sensory attributes of cladodes. Therefore, the objective was to estimate the pyruvate decarboxylase activity in young cladodes of 'Tiny' palms - Nopalea cochenilifera and 'Ear Mexican Elephant' - Opuntia tuna minimally processed. The experiment was conducted in the Laboratory of Chemistry, Biochemistry and Food Analysis Center of Science and Technology Agrifood the Federal University of Campina Grande, Campus Pombal, Paraíba. The young cladodes were minimally processed and the analyzes were performed immediately after processing, with 24 and 48 hours of incubation under controlled temperature (22 ± 2°C). The analyzes performed were activity of the enzyme pyruvate decarboxylase (PDC), pH, soluble solids, titratable acidity, ratio, soluble sugars, total chlorophyll, total carotenoids, ascorbic acid and phenolic compounds. There was enzymatic activity of pyruvate decarboxylase from young minimally processed cladodes for the species studied, with greater activity in the cladodes of the species 'Tiny'.

Foliar treatment of potassium nitrate modulates the fermentative and sucrose metabolizing pathways in contrasting maize genotypes under water logging stress.

The effect of potassium nitrate on the status of fermentative and sucrose metabolizing pathways was studied in two maize (Zea mays L.) genotypes, viz., LM 5 (relatively susceptible to flooding) and I 167 (relatively tolerant to flooding) under water logging stress. The higher increase in pyruvate decarboxylase, alcohol dehydrogenase and aldehyde dehydrogenase activities in the hypoxic roots of I 167 seedlings over LM 5 showed the former's efficient tolerance mechanism towards anaerobic conditions. Foliar application of KNO3 reduced these enzymatic activities in the roots of both the genotypes. The shoots of I 167 seedlings also showed a parallel increase in alcohol dehydrogenase and pyruvate decarboxylase activities under water logging stress. These enzymatic activities, however, remained unaffected in shoots of water logged LM 5 seedlings. There was a higher decrease in acid and alkaline invertase activities in the hypoxic roots of I 167 seedlings. KNO3 treatment led to higher acid invertase activity in roots of I 167 seedlings than those of LM 5. Sucrose synthase (synthesis) and sucrose phosphate synthase activities decreased, but sucrose synthase (breakdown) activity increased in the roots of both the genotypes, during water logging. KNO3 increased sucrose synthesizing activities with a parallel increase in the sucrose content of the roots. Sucrose synthesis was comparatively unaffected in I 167 shoots under water logging stress while LM 5 shoots showed higher reduction in its sucrose synthase (synthesis) and sucrose phosphate synthase activities. It may thus be concluded that KNO3 induced a network of reactions for improving water logging tolerance. The nitrate ions acted as an alternate electron acceptor and thus reduced the activities of fermentative enzymes. It promoted the funneling of sugars into the glycolytic pathway by inducing the activities of acid and alkaline invertases in the roots and shoots of maize genotypes. It also directed the hexoses towards biosynthetic pathway by increasing the activities of sucrose synthesizing enzymes.

Pretreatment with Trichoderma harzianum alleviates waterlogging-induced growth alterations in tomato seedlings by modulating physiological, biochemical, and molecular mechanisms

We studied the role of Trichoderma harzianum (TH) in improving the physiological, biochemical, hormonal, and molecular parameters of tomato seedlings grown under waterlogging (WL, for 14 and 28 days). Pretreatment with TH significantly improved the growth of tomato by enhancing the chlorophyll synthesis and uptake of essential ions, including nitrogen, phosphorus, and potassium. A reduction in anthocyanin content was also ameliorated significantly by TH pretreatment. TH significantly mitigated the WL-induced decline in height and in fresh and dry biomass accumulation. Accumulation of proline, flavonoids, anthocyanin, sugars, and soluble protein increased with TH pretreatment. At both growth periods (14 and 28 days after treatment [DAT]), the accumulation of secondary metabolites, including total phenols and flavonoids, and the redox components (tocopherols) were increased significantly by TH pretreatment. Increased synthesis of metabolites maintained the antioxidant status of tomato, resulting in amelioration of WL-induced oxidative effects on membranes. WL and TH treatments significantly increased ethylene production and decreased abscisic acid content at both growth periods. The accumulation of reactive oxygen species, like hydrogen peroxide, in TH treated seedlings was correlated with the upregulation of the Fe-SOD gene. WL stress triggered the activity of sucrose synthase (SUS), lactate dehydrogenase (LDH), and pyruvate decarboxylase (PDC), which reached a maximum at 14 DAT, and TH pretreatment resulted in further enhancement above control and WL-stressed levels. Quantitative RT-PCR revealed differential expression of genes, where Fe-SOD and ADH were upregulated due to TH treatment and ARE, ACO, ERF, and aquaporin were downregulated relative to control plants. Pretreatment of tomato seedlings with TH improved tolerance to WL by maintaining the antioxidant status, sugar metabolism, and expression of critical genes. These results suggest that TH pretreatment is an effective way to improve WL tolerance in tomato at vegetative stage.

Batch and continuous cultivation processes of Candida tropicalis TISTR 5306 for ethanol and pyruvate decarboxylase production in fresh longan juice with optimal carbon to nitrogen molar ratio

AbstractThe carbon to nitrogen (C/N) molar ratio of 21.88 ± 0.20 in fresh juice from C‐grade longan (FLJ‐C) supplemented with ammonium sulfate (AMS) was optimal for cultivation of Candida tropicalis TISTR 5306 producing ethanol concentration of 24.0 ± 1.1 g/L and specific pyruvate decarboxylase (PDC) activity of 0.138 ± 0.001 U/mg protein. The highest ethanol concentration in a 10 L continuous process (34.3 ± 0.5 g/L, dilution rate [D] = 0.0492 hr−1) was significantly higher (p ≤ .05) than that in 100 L batch process (13.2 ± 0.2 g/L at 108 hr). The highest volumetric PDC activities of 0.081 ± 0.001 U/ml (D = 0.0070 hr−1) and 0.107 ± 0.001 U/ml (48 hr) were attained for continuous and batch processes, respectively. The affinity constants and growth‐related weighting factors on sucrose and monosaccharides (glucose and fructose with the same values) utilization were (123 ± 1 g/L, 0.14 ± 0.01) and (34.1 ± 1.5 g/L, 0.43 ± 0.01) with MS and R2 of 92.2 and .9552, respectively.Practical applicationsFLJ‐C is an example of agricultural produces with relatively high production quantity (50,000 t/year in Thailand) and high sugars content but low marketing demands due to the smallest size of pulp. The complex nitrogen source contains costly components (yeast extract—78.3 USD/kg, malt extract—148 USD/kg, and peptone—151 USD/kg) which can be replaced with an inexpensive AMS (1.05 USD/kg). Further consideration of C/N molar ratio in FLJ‐C can optimize growth, ethanol (alternative biofuel/industrial solvent) production, and specific PDC (biocatalyst for phenylacetylcarbinol—PAC, anti‐asthmatic and nasal decongestant precursors) activity of C. tropicalis TISTR 5306 whole cells which can be used as a case study for other related yeasts. Batch and continuous production at 100 L and 10 L also illustrate practical utilization of FLJ‐C with AMS supplement at larger scales for development of subsequent zero waste process.

The Transcriptome and Flux Profiling of Crabtree-Negative Hydroxy Acid-Producing Strains of Saccharomyces cerevisiae Reveals Changes in the Central Carbon Metabolism.

Saccharomyces cerevisiae (S. cerevisiae) is the yeast cell factory of choice for the production of many biobased chemicals. However, it is a Crabtree-positive yeast and so shuttles a large portion of carbon into ethanol. Ethanol formation can be eliminated by deleting pyruvate decarboxylase (PDC) activity. It is not yet well understood how PDC-negative yeasts are affected when engineered to produce other products than ethanol. In this study, pathways are introduced for the production of three hydroxy acids (lactic, malic, or 3-hydroxypropionic acid [3HP]) into an evolved PDC-negative strain. These strains are characterized via transcriptome and flux profiling to elucidate the effects that the production of these hydroxy acids has on the host strain. Expression of lactic and malic acid biosynthesis pathways improved the maximum specific growth rate (μmax ) of the strain by 64% and 20%, respectively, presumably due to nicotinamide adenine dinucleotide regeneration. All strains show a very high flux ( > 90% of glucose uptake) into the oxidative pentose phosphate pathway under batch fermentation conditions. The study, for the first time, directly compares the flux and transcriptome profiles of several hydroxy acid-producing strains of an evolved PDC-negative S. cerevisiae and suggests directions for future metabolic engineering.

AdRAP2.3, a Novel Ethylene Response Factor VII from Actinidia deliciosa, Enhances Waterlogging Resistance in Transgenic Tobacco through Improving Expression Levels of PDC and ADH Genes.

APETALA2/ethylene-responsive factor superfamily (AP2/ERF) is a transcription factor involved in abiotic stresses, for instance, cold, drought, and low oxygen. In this study, a novel ethylene-responsive transcription factor named AdRAP2.3 was isolated from Actinidia deliciosa ‘Jinkui’. AdRAP2.3 transcription levels in other reproductive organs except for the pistil were higher than those in the vegetative organs (root, stem, and leaf) in kiwi fruit. Plant hormones (Salicylic acid (SA), Methyl-jasmonate acid (MeJA), 1-Aminocyclopropanecarboxylic Acid (ACC), Abscisic acid (ABA)), abiotic stresses (waterlogging, heat, 4 °C and NaCl) and biotic stress (Pseudomonas Syringae pv. Actinidiae, Psa) could induce the expression of AdRAP2.3 gene in kiwi fruit. Overexpression of the AdRAP2.3 gene conferred waterlogging stress tolerance in transgenic tobacco plants. When completely submerged, the survival rate, fresh weight, and dry weight of transgenic tobacco lines were significantly higher than those of wile type (WT). Upon the roots being submerged, transgenic tobacco lines grew aerial roots earlier. Overexpression of AdRAP2.3 in transgenic tobacco improved the pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) enzyme activities, and improved the expression levels of waterlogging mark genes NtPDC, NtADH, NtHB1, NtHB2, NtPCO1, and NtPCO2 in roots under waterlogging treatment. Overall, these results demonstrated that AdRAP2.3 might play an important role in resistance to waterlogging through regulation of PDC and ADH genes in kiwi fruit.

Conservation of ethanol fermentation and its regulation in land plants.

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.

Relationship between Polyamines and Anaerobic Respiration of Wheat Seedling Root under Water-Logging Stress

To elucidate the relationship between polyamines and anaerobic respiration of wheat (Triticum aestivum L.) seedling root under water-logging stress, the contents of polyamines (PAs), lactate and alcohol, and the activities of anaerobic respiration enzymes were investigated in seedling roots of two wheat cultivars, Yumai no. 18 and Yangmai no. 9. On the 5th day after water-logging treatment, spermidine (Spd) and spermine (Spm) contents increased significantly, pyruvate decarboxylase (PDC) activity increased and there was no difference between two cultivars. Alcohol dehydrogenase (ADH) activity and alcohol content in Yangmai no. 9 increased more markedly than Yumai no. 18, while lactate dehydrogenase (LDH) activity and the lactate content in the Yumai no. 18 increased more markedly than Yangmai no. 9. Treatments with exogenous Spd and Spm resulted in enhancing the increases in ADH activity, alcohol content, and the levels of Spd and Spm. This concomitantly inhibited the increases in LDH activity and lactate content in Yumai no. 18 under water-logging stress, alleviating stress-induced injury to the seedlings. Treatment with exogenous inhibitor methylglyoxyl-bis-guanylhydrazone (MGBG), resulted in reducing the increases in ADH activity, alcohol content, and Spd and Spm levels, promoting the increases in LDH activity and lactate content in Yangmai no. 9 under water-logging stress, and aggravating the stress-induced injury to the seedlings. The results suggested that under water-logging stress, increased Spd and Spm could facilitate the tolerance of wheat seedling to the stress by enhancing the increases in ADH activity and alcohol content, and inhibiting the increases in LDH activity and lactate content.

Acetaldehyde and Ethanol Metabolism during Conditioning and Air Storage of ‘Honeycrisp’ Apples

‘Honeycrisp’ apples are susceptible to the physiological disorder soft scald, especially when stored at temperatures close to 0 °C. The disorder can be reduced by a conditioning treatment of 10 °C for 7 days before storage, but little is known about the underlying physiology of disorder development. The effects of storing ‘Honeycrisp’ apples in air at 0.5 °C for a total of 140 days, without and with conditioning, on internal ethylene concentration (IEC), ethanol and acetaldehyde concentrations, and activities of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) were investigated in relation to soft scald incidence. Fruit were selected on the basis of background color (chlorophyll concentration) using a nondestructive delta absorbance (DA) meter to minimize variability of fruit maturation. Conditioning reduced soft scald incidence to 1% compared with 28% in unconditioned fruit. During the conditioning period, IECs were usually greatest in the conditioned fruit, with no effect on ethanol and acetaldehyde concentrations. During subsequent storage, IEC was greatest in conditioned fruit, whereas ethanol and acetaldehyde concentrations were generally less. However, ADH and PDC activities were unaffected consistently by conditioning or during storage.

Bioconversion of hemicellulosic materials into ethanol by yeast, Pichia kudriavzevii 2-KLP1, isolated from industrial waste

In the present work, a yeast strain Pichia kudriavzevii was identified on the basis of 18S rDNA, showing maximum growth at 30°C and pH 7.0. Among all the complex polysaccharides used, wheat bran proved to be the best substrate as indicated by the maximum growth of the yeast strain. The yeast isolate was capable of producing xylanase both intra- and extra-cellularly, the dominant form being extracellular. The maximum enzyme activity was determined at pH 5.0 and at 50°C. Na+, Mg2+ and Fe2+ presence caused a substantial increase in enzyme activity while a slight decrease (4.5%) was observed in the presence of Mn2+, Zn2+ and Cu2+. Pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) activities were assayed to confirm the presence of the ethanol pathway and PDC activity was much more pronounced (73%) compared to ADH activity (51%). The yeast strain can be employed to utilize hemicellulose containing agroindustrial residues for ethanol production.

Transcriptomic and physiological studies identify cues for germination stage oxygen deficiency tolerance in rice

Submergence during germination imposes germination stage oxygen deficiency (GSOD). Natural variation in anaerobic germination potential (AGP), manifested by their differences in GSOD tolerance, is available in rice. Present study was performed to understand differential responses of two rice cultivars, Naveen and AC41620 (susceptible and tolerant to GSOD, respectively) subjecting them to 48-h continuous submergence during germination. Transcriptome analysis revealed more elaborate regulation of gene expression in AC41620 compared to Naveen. Validation of transcriptome data through q-PCR of selected genes and biochemical analysis related to carbohydrate metabolism, anaerobic respiration and oxidative stress tolerance were more significant in AC41620, than Naveen. The time-course enzyme assays for continued submergence suggested that the activity of α-amylase, pyruvate decarboxylase and alcohol dehydrogenase increased under continuous submergence. Efficient nitrogen metabolism supported by increased nitrate and nitrite reductase activities and better handling of reactive oxygen species (ROS) because of increased antioxidant enzyme activities were found to be associated with GSOD in AC41620. Increased ethylene production along with action of ABA, GA and G-proteins aided in GSOD tolerance of AC41620. The differentially expressed genes identified in the present study will serve as a useful genomic resource for extending our understanding of molecular mechanism of anaerobic germination vis-à-vis GSOD tolerance in rice.

Kinetic modelling: an integrated approach to analyze enzyme activity assays

BackgroundIn general, enzyme activity is estimated from spectrophotometric data, by taking the slope of the linear part of the progress curve describing the rate of change in the substrate or product monitored. As long as the substrate concentrations are sufficiently high to saturate the enzyme and, the velocity of the catalyzed reaction is directly proportional to the enzyme concentration. Under these premises, this velocity can be taken as a measure of the amount of active enzyme present. Estimation of the enzyme activity through linear regression of the data should only be applied when linearity is true, which is often not the case or has not been checked.ResultsIn this paper, we propose a more elaborate method, based on a kinetic modelling approach, to estimate the in vitro specific enzyme activity from spectrophotometric assay data. As a case study, kinetic models were developed to estimate the activity of the enzymes pyruvate decarboxylase and alcohol dehydrogenase extracted from ‘Jonagold’ apple (Malus x domestica Borkh. cv. ‘Jonagold’). The models are based on Michaelis–Menten and first order kinetics, which describe the reaction mechanism catalyzed by the enzymes.ConclusionsIn contrast to the linear regression approach, the models can be used to estimate the enzyme activity regardless of whether linearity is achieved since they integrally take into account the complete progress curve. The use of kinetic models to estimate the enzyme activity can be applied to all other enzymes as long as the underlying reaction mechanism is known. The kinetic models can also be used as a tool to optimize the enzyme assays by systematically studying the effect of the various design parameters.

Seed Pelleting with Calcium Peroxide Improves Crop Establishment of Direct-seeded Rice under Waterlogging Conditions

Poor crop establishment of direct-seeded rice (DSR) is one of the major constraints to wide adoption of DSR, particularly in areas prone to flooding after sowing or where fields are not level. Seed pelleting is an effective, practical and facile technique to enhance crop establishment under unfavorable environmental conditions. To evaluate the effects of seed pelleting on rice germination, seedling growth and associated metabolic events under waterlogging stress, various seed pelleting treatments including formulae, pelleting times (the weight ratio of pelleting agents: rice seeds = 1:1~7:1 (w/w) and CaO2 contents were tested in series of experiments. Naked seeds were maintained for comparison as a control. Pelleting treatments with CaO2 significantly increased seed germination and seedling growth of DSR under waterlogging conditions compared with pelleted seeds without CaO2 and naked seeds. The optimum weight ratios of CaO2 to dry seeds were found to be in the range of 0.6:1–1:1 based on seed germination and seedling growth performance under waterlogging conditions. Under waterlogging conditions, high seed germination percentage and vigorous seedling growth of DSR due to seed pelleting with CaO2 was associated with an increase in α-amylase activity, but decrease in alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) activities in pelleted seeds.

Proteomic Analysis of Calcium Effects on Soybean Root Tip under Flooding and Drought Stresses.

Flooding and drought are disadvantageous environmental conditions that induce cytosolic calcium in soybean. To explore the effects of flooding- and drought-induced increases in calcium, a gel-free/label-free proteomic analysis was performed. Cytosolic calcium was decreased by blocking calcium channels in the endoplasmic reticulum (ER) and plasma membrane under both stresses. Calnexin, protein disulfide isomerase, heat shock proteins and thioredoxin were predominantly affected as the ER proteins in response to calcium, and ER-associated degradation-related proteins of HCP-like superfamily protein were up-regulated under stress exposure and then down-regulated. Glycolysis, fermentation, the tricarboxylic acid cycle and amino acid metabolism were mainly induced as the types of cellular metabolism in response to calcium under both stresses. Pyruvate decarboxylase was increased and decreased under flooding and drought, respectively, and was further decreased by the reduction of cytosolic calcium; however, it was recovered by exogenous calcium under both stresses. Furthermore, pyruvate decarboxylase activity was increased under flooding, but decreased under drought. These results suggest that calcium is involved in protein folding in the ER, and ER-associated degradation might alleviate ER stress during the early stage of both stresses. Furthermore, calcium appears to modify energy metabolism, and pyruvate decarboxylase may be a key enzyme in this process under flooding and drought.

Dietary manganese requirement of juvenile hybrid grouper, Epinephelus lanceolatus × E. fuscoguttatus

An 8-week feeding trial was conducted to estimate the optimum dietary manganese (Mn) requirement for juvenile hybrid grouper, Epinephelus lanceolatus × E. fuscoguttatus. The basal diet was formulated to contain 520 g/kg crude protein from casein and fishmeal. Manganese methionine was added to the basal diet at 0 (control group), 2.5, 5, 10, 20 and 40 mg Mn/kg diet providing 7.48, 10.34, 13.76, 19.72, 31.00 and 53.91 mg Mn/kg diet, respectively. Each diet was randomly fed to triplicate groups of juveniles, and each tank was stocked with 20 fish (initial weight, 60.06 ± 0.68 g). The manganese content in rearing water was monitored and kept below 0.01 mg/L. Results showed that the weight gain ratio (WGR), protein efficiency ratio (PER), specific growth rate (SGR), Mn contents in whole body, liver and vertebra, and activities of hepatic Mn superoxide dismutase (Mn-SOD), total SOD (T-SOD) and glutathione peroxidase (GSH-PX) were significantly improved by dietary Mn supplementation (p < .05). However, dietary Mn did not affect arginase (DArg) activity. The highest feed conversion ratio (FCR) was observed in fish fed the basal diet (p < .05). No significant differences were found on the Cu and Zn contents in whole body by supplementing dietary Mn. Supplemented Mn in diets had significantly effect on liver and vertebral trace element deposition (p < .05). Fish fed the basal diet had the highest Fe and Zn contents in vertebra (p < .05). There were no significant differences on hepatic pyruvate decarboxylase (PDC) activity with supplemented Mn levels below 13.76 mg/kg. As biomarker of oxidative stress, malondialdehyde (MDA) content in liver was significantly higher in fish fed the basal diet (p < .05). Using the broken-line models based on SGR, dietary Mn requirement of the juvenile hybrid grouper was estimated to be 12.70 mg/kg diet.

Significance of Individual Residues at the Regulatory Site of Yeast Pyruvate Decarboxylase for Allosteric Substrate Activation

The catalytic activity of the allosteric enzyme pyruvate decarboxylase from yeast is strictly controlled by its own substrate pyruvate via covalent binding at a separate regulatory site. Kinetic studies, chemical modifications, cross-linking, small-angle X-ray scattering, and crystal structure analyses have led to a detailed understanding of the substrate activation mechanism at an atomic level with C221 as the core moiety of the regulatory site. To characterize the individual role of the residues adjacent to C221, we generated variants H92F, H225F, H310F, A287G, S311A, and C221A/C222A. The integrity of the protein structure of the variants was established by small-angle X-ray scattering measurements. The analyses of both steady state and transient kinetic data allowed the identification of the individual roles of the exchanged side chains during allosteric enzyme activation. In each case, the kinetic pattern of activation was modulated but not completely abolished. Despite the crucial role of C221, the covalent binding of pyruvate is not obligate for enzyme activation but is a requirement for a kinetically efficient transition from the inactive to the active state. Moreover, only one of the three histidines guiding the activator molecule to the binding pocket, H310, specifically interacts with C221. H310 stabilizes the thiolate form of C221, ensuring a rapid nucleophilic attack of the thiolate sulfur on C2 of the regulatory pyruvate, thus forming a regulatory dyad. The influence of the other two histidines is less pronounced. Substrate activation is slightly weakened for A287G and significantly retarded for S311A.

Improvement of whole-cell transamination with Saccharomyces cerevisiae using metabolic engineering and cell pre-adaptation

BackgroundWhole-cell biocatalysis based on metabolically active baker’s yeast with engineered transamination activity can be used to generate molecules carrying a chiral amine moiety. A prerequisite is though to express efficient ω-transaminases and to reach sufficient intracellular precursor levels.ResultsHerein, the efficiency of three different ω-transaminases originating from Capsicum chinense, Chromobacterium violaceum, and Ochrobactrum anthropi was compared for whole-cell catalyzed kinetic resolution of racemic 1-phenylethylamine to (R)-1-phenylethylamine. The gene from the most promising candidate, C. violaceum ω-transaminase (CV-TA), was expressed in a strain lacking pyruvate decarboxylase activity, which thereby accumulate the co-substrate pyruvate during glucose assimilation. However, the conversion increased only slightly under the applied reaction conditions. In parallel, the effect of increasing the intracellular pyridoxal-5′-phosphate (PLP) level by omission of thiamine during cultivation was investigated. It was found that without thiamine, PLP supplementation was redundant to keep high in vivo transamination activity. Furthermore, higher reaction rates were achieved using a strain containing several copies of CV-TA gene, highlighting the necessity to also increase the intracellular transaminase level. At last, this strain was also investigated for asymmetric whole-cell bioconversion of acetophenone to (S)-1-phenylethylamine using l-alanine as amine donor. Although functionality could be demonstrated, the activity was extremely low indicating that the native co-product removal system was unable to drive the reaction towards the amine under the applied reaction conditions.ConclusionsAltogether, our results demonstrate that (R)-1-phenylethylamine with >99% ee can be obtained via kinetic resolution at concentrations above 25 mM racemic substrate with glucose as sole co-substrate when combining appropriate genetic and process engineering approaches. Furthermore, the engineered yeast strain with highest transaminase activity was also shown to be operational as whole-cell catalyst for the production of (S)-1-phenylethylamine via asymmetric transamination of acetophenone, albeit with very low conversion.

Calcium treatments promote the aroma volatiles emission of pear (Pyrus ussuriensis ‘Nanguoli’) fruit during post-harvest ripening process

Aroma is an important factor affecting pear fruit quality. This study was undertaken to assess whether pre-harvest calcium sprays (15 d before harvest, applications at 4%, w/v, 120 d after full bloom) could improve aroma of Pyrus ussuriensis ‘Nanguoli’ pear fruit at harvest and post-harvest, and analyse the mechanism of metabolic regulation.Most compounds contributing to overall flavor in ripe ‘Nanguoli’ fruit at harvest and post-harvest were enhanced in response to pre-harvest calcium applications. Ester, especially acetyl ester, content was enhanced by calcium treatment; however, contents of alcohols, aldehydes and hydrocarbons were little affected. These effects were consistent with higher pyruvate decarboxylase and alcohol dehydrogenase activities in treated fruit, suggesting that calcium treatment increased supply of alcohols and acyl CoAs for ester biosynthesis. We also found that activities of β-glucosidases (involved in the emission of bound volatile compounds) were improved and the contents of bound volatiles were reduced by calcium treatment. At the same time, relative contents of linoleic and linolenic acids were improved by calcium treatment, expression of genes encoding fatty acid desaturase involved in the biosynthesis of unsaturated fatty acid precursors (linoleic and linolenic acids) for volatile aroma were improved. Calcium treatment increased content of volatile aromatic substances, both because calcium promoted the decomposition of bound aroma substances to free aromatic forms, and also promoted the synthesis of volatile aromatic substances in pear fruit.

Enhanced production of 2,3-butanediol by engineered Saccharomyces cerevisiae through fine-tuning of pyruvate decarboxylase and NADH oxidase activities.

Background2,3-Butanediol (2,3-BD) is a promising compound for various applications in chemical, cosmetic, and agricultural industries. Pyruvate decarboxylase (Pdc)-deficient Saccharomyces cerevisiae is an attractive host strain for producing 2,3-BD because a large amount of pyruvate could be shunted to 2,3-BD production instead of ethanol synthesis. However, 2,3-BD yield, productivity, and titer by engineered yeast were inferior to native bacterial producers because of the following metabolic limitations. First, the Pdc-deficient yeast showed growth defect due to a shortage of C2-compounds. Second, redox imbalance during the 2,3-BD production led to glycerol formation that lowered the yield.ResultsTo overcome these problems, the expression levels of Pdc from a Crabtree-negative yeast were optimized in S. cerevisiae. Specifically, Candida tropicalis PDC1 (CtPDC1) was used to minimize the production of ethanol but maximize cell growth and 2,3-BD productivity. As a result, productivity of the BD5_G1CtPDC1 strain expressing an optimal level of Pdc was 2.3 folds higher than that of the control strain in flask cultivation. Through a fed-batch fermentation, 121.8 g/L 2,3-BD was produced in 80 h. NADH oxidase from Lactococcus lactis (noxE) was additionally expressed in the engineered yeast with an optimal activity of Pdc. The fed-batch fermentation with the optimized 2-stage aeration control led to production of 154.3 g/L 2,3-BD in 78 h. The overall yield of 2,3-BD was 0.404 g 2,3-BD/g glucose which corresponds to 80.7% of theoretical yield.ConclusionsA massive metabolic shift in the engineered S. cerevisiae (BD5_G1CtPDC1_nox) expressing NADH oxidase was observed, suggesting that redox imbalance was a major bottleneck for efficient production of 2,3-BD by engineered yeast. Maximum 2,3-BD titer in this study was close to the highest among the reported microbial production studies. The results demonstrate that resolving both C2-compound limitation and redox imbalance is critical to increase 2,3-BD production in the Pdc-deficient S. cerevisiae. Our strategy to express fine-tuned PDC and noxE could be applicable not only to 2,3-BD production, but also other chemical production systems using Pdc-deficient S. cerevisiae.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0677-9) contains supplementary material, which is available to authorized users.

Mechanistic and Structural Insight to an Evolved Benzoylformate Decarboxylase with Enhanced Pyruvate Decarboxylase Activity

Benzoylformate decarboxylase (BFDC) and pyruvate decarboxylase (PDC) are thiamin diphosphate-dependent enzymes that share some structural and mechanistic similarities. Both enzymes catalyze the nonoxidative decarboxylation of 2-keto acids, yet differ considerably in their substrate specificity. In particular, the BFDC from P. putida exhibits very limited activity with pyruvate, whereas the PDCs from S. cerevisiae or from Z. mobilis show virtually no activity with benzoylformate (phenylglyoxylate). Previously, saturation mutagenesis was used to generate the BFDC T377L/A460Y variant, which exhibited a greater than 10,000-fold increase in pyruvate/benzoylformate substrate utilization ratio compared to that of wtBFDC. Much of this change could be attributed to an improvement in the Km value for pyruvate and, concomitantly, a decrease in the kcat value for benzoylformate. However, the steady-state data did not provide any details about changes in individual catalytic steps. To gain insight into the changes in conversion rates of pyruvate and benzoylformate to acetaldehyde and benzaldehyde, respectively, by the BFDC T377L/A460Y variant, reaction intermediates of both substrates were analyzed by NMR and microscopic rate constants for the elementary catalytic steps were calculated. Herein we also report the high resolution X-ray structure of the BFDC T377L/A460Y variant, which provides context for the observed changes in substrate specificity.