Pyruvate Carboxylase Activity Research Articles

MafA-positive insulin-producing cells derived from rat adipose stem cells are efficient in glucose hemostasis in vitro

Objective: MafA has been recognized as a specific pancreatic β-cells activator for Insulin expression. This study investigated the changes in glucose transport and consumption by MafA-positive Insulin-producing cells resulting from the differentiation of rat adipose tissue-derived mesenchymal stem cells. Methods: Adipose tissue-derived mesenchymal stem cells were differentiated into Insulin-producing cells in a 14-day protocol using nicotinamide and ITS. The ability to produce Insulin was confirmed by adipogenesis differentiation capacity and expression of Oct4, Nanog, and Sox2. Insulin and Glucagon concentrations in Insulin-producing cells were determined by ELISA. The ability of Insulin-producing cells for the presence of MafA, Glut2, ENO1, and IGF-I receptor genes was determined by Western blotting analysis. The amounts of Glucokinase and Pyruvate Carboxylase activities were measured by Fluorometric and Colorimetric methods, respectively. Results: The un-differentiated cells showed a promising capacity to differentiate toward adipocytes. The adipose tissue mesenchymal stem cells revealed significantly elevated levels of Oct4, Nanog, and Sox2 compared to differentiated adipocytes. The cells that differentiated into Insulin-producing cells secreted considerably more Insulin and Glucagon than undifferentiated cells.

Resveratrol biotransformation and actions on the liver metabolism of healthy and arthritic rats

Aimsto investigate the effects of resveratrol on glycogen catabolism and gluconeogenesis in perfused livers of healthy and arthritic rats. The actions of resveratrol-3-O-glucuronide (R3G) and the biotransformation of resveratrol into R3G was further evaluated in the livers. Main methodsarthritis was induced with Freund's adjuvant. Resveratrol at concentrations of 10, 25, 50, 100 and 200 μM and 200 μM R3G were introduced in perfused livers. Resveratrol and metabolites were measured in the outflowing perfusate. Respiration of isolated mitochondria and activity of gluconeogenic enzymes were also evaluated in the livers. Key findingsresveratrol inhibited glycogen catabolism when infused at concentrations above 50 μM and gluconeogenesis even at 10 μM in both healthy and arthritic rat livers, but more sensitive in these latter. Resveratrol above 100 μM inhibited ADP-stimulated respiration and the activities of NADH- and succinate-oxidases in mitochondria, which were partially responsible for gluconeogenesis inhibition. Pyruvate carboxylase activity was inhibited by 25 μM resveratrol and should inhibit gluconeogenesis already at low concentrations. Resveratrol was significantly metabolized to R3G in healthy rat livers, however, R3G formation was lower in arthritic rat livers. The latter must be in part a consequence of a lower glucose disposal for glucuronidation. When compared to resveratrol, R3G inhibited gluconeogenesis in a lower extension and glycogen catabolism in a higher extension. Significancethe effects of resveratrol and R3G tended to be transitory and existed only when the resveratrol is present in the organ, however, they should be considered because significant serum concentrations of both are found after oral ingestion of resveratrol.

Enhancement of Carbon Assimilates and Macronutrients in Legumes under Elevated CO2 Concentration

Impact of elevated carbon dioxide (Free Air Carbon dioxide Enrichment) was studied on the plant chlorophyll, plant growth, plant macronutrients, total starch, total carbohydrate and the activity of phosphoenol pyruvate carboxylase (Pep C) enzymatic assays in leaves, pods and seeds in leguminous plant Cyamopsis tetragonoloba. Plants of Cyamopsis tetragonoloba (C3) were exposed to different atmospheric CO2 concentrations 420 ± 20 ppm (ambient) and 550 ± 20ppm (elevated). An average increase in the plant total chlorophyll (+39.17%), total starch (+43.73%, +25.44% and +26.35% in leaves, pods, and seeds), sucrose (+69.77%, +22.27% and +33.77% in leaves, pods and seed) and total carbohydrate (+58.88%, +30.54% and +28.38 % in leaves, pods and seeds) content were found in plant grown under elevated condition when compared to ambient counterpart. More over overall plant growth (+40% height and +25% biomass) increased in [CO2] concentration. Plant total nitrogen (N) content decreased (−12.55% in leaves) under the elevated condition where as total phosphorus (P) decreased (−3.15% in leaves) along with total potassium (K) (−46.63% in leaves). In soil, total potassium (+60.23%) and phosphorous (+48.88%) were found to increase with (−16%) decrease in soil nitrogen content. In seed total nitrogen content increased (+18.15%) on an average with no significant change in total potassium and phosphorus content under e[CO2]. Phosphoenolpyruvate carboxylase enzyme (Pep C) (+139.5% in leaves) activity and total organic carbon (TOC) (+19.12%, +17.85% in leaves and seeds) increases in elevated concentration thus promoting and indicating higher photosynthesis via enhanced CO2 fixation. Thus our studies showed that [CO2] positively promotes sugars, carbohydrates synthesis, translocation and partitioning in plant tissues and enhanced macronutrients level in leaves and seeds tissue which is contradictory to other C3 plants. Thus [CO2] works as the boon for Cyamopsisvarieties and the seeds are nutritionally rich, healthy, balanced in proteins and carbohydrates (C/N) and so these varieties have future implication for industrial use in the agricultural country like India.

1α,25-dihydroxyvitamin D reduction of MCF10A-ras cell viability in extracellular matrix detached conditions is dependent on regulation of pyruvate carboxylase

An emerging hallmark of cancer is cellular metabolic reprogramming to adapt to varying cellular environments. Throughout the process of metastasis cancer cells gain anchorage independence which confers survival characteristics when detached from the extracellular matrix (ECM). Previous work demonstrates that the bioactive metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25[OH]2D), suppresses cancer progression, potentially by suppressing the ability of cells to metabolically adapt to varying cellular environments such as ECM detachment. The purpose of the present study was to determine the mechanistic bases of the effects of 1,25(OH)2D on cell survival in ECM-detached conditions. Pretreatment of MCF10A-ras breast cancer cells for 3 d with 1,25(OH)2D reduced the viability of cells in subsequent detached conditions by 11%. Enrichment of 13C5-glutamine was reduced in glutamate (21%), malate (30%), and aspartate (23%) in detached compared to attached MCF10A-ras cells. Pretreatment with 1,25(OH)2D further reduced glutamine flux into downstream metabolites glutamate (5%), malate (6%), and aspartate (10%) compared to detached vehicle treated cells. Compared to attached cells, detachment increased pyruvate carboxylase (PC) mRNA abundance and protein expression by 95% and 190%, respectively. Consistent with these results, 13C6-glucose derived M+3 labelling was shown to preferentially replenish malate and aspartate, but not citrate pools, demonstrating increased PC activity in detached cells. In contrast, 1,25(OH)2D pretreatment of detached cells reduced PC mRNA abundance and protein expression by 63% and 56%, respectively, and reduced PC activity as determined by decreased 13C6-glucose derived M+3 labeling in citrate (8%) and aspartate (50%) pools, relative to vehicle-treated detached cells. While depletion of PC with doxycycline-inducible shRNA reduced detached cell viability, PC knockdown in combination with 1,25(OH)2D treatment did not additionally affect the viability of detached cells. Further, PC overexpression improved detached cell viability, and inhibited the effect of 1,25(OH)2D on detached cell survival, suggesting that 1,25(OH)2D mediates its effects in detachment through regulation of PC expression. These results suggest that inhibition of PC by 1,25(OH)2D suppresses cancer cell anchorage independence.

A multidimensional metabolomics workflow to image biodistribution and evaluate pharmacodynamics in adult zebrafish.

ABSTRACTAn integrated evaluation of the tissue distribution and pharmacodynamic properties of a therapeutic is essential for successful translation to the clinic. To date, however, cost-effective methods to measure these parameters at the systems level in model organisms are lacking. Here, we introduce a multidimensional workflow to evaluate drug activity that combines mass spectrometry-based imaging, absolute drug quantitation across different biological matrices, in vivo isotope tracing and global metabolome analysis in the adult zebrafish. As a proof of concept, we quantitatively determined the whole-body distribution of the anti-rheumatic agent hydroxychloroquine sulfate (HCQ) and measured the systemic metabolic impacts of drug treatment. We found that HCQ distributed to most organs in the adult zebrafish 24 h after addition of the drug to water, with the highest accumulation of both the drug and its metabolites being in the liver, intestine and kidney. Interestingly, HCQ treatment induced organ-specific alterations in metabolism. In the brain, for example, HCQ uniquely elevated pyruvate carboxylase activity to support increased synthesis of the neuronal metabolite, N-acetylaspartate. Taken together, this work validates a multidimensional metabolomics platform for evaluating the mode of action of a drug and its potential off-target effects in the adult zebrafish. This article has an associated First Person interview with the first author of the paper.

Tumor cells dictate anti-tumor immune responses by altering pyruvate utilization and succinate signaling in CD8+ T cells

The tumor microenvironment (TME) is a unique metabolic niche that can inhibit Tcell metabolism and cytotoxicity. To dissect the metabolic interplay between tumors and Tcells, we establish an invitro system that recapitulates the metabolic niche of the TME and allows us to define cell-specific metabolism. We identify tumor-derived lactate as an inhibitor of CD8+ Tcell cytotoxicity, revealing an unexpected metabolic shunt in the TCA cycle. Metabolically fit cytotoxic Tcells shunt succinate out of the TCA cycle to promote autocrine signaling via the succinate receptor (SUCNR1). Cytotoxic Tcells are reliant on pyruvate carboxylase (PC) to replenish TCA cycle intermediates. By contrast, lactate reduces PC-mediated anaplerosis. The inhibition of pyruvate dehydrogenase (PDH) is sufficient to restore PC activity, succinate secretion, and the activation of SUCNR1. These studies identify PDH as a potential drug target to allow CD8+ Tcells to retain cytotoxicity and overcome a lactate-enriched TME.

Effects of Rumen-Protected Creatine Pyruvate on Meat Quality, Hepatic Gluconeogenesis, and Muscle Energy Metabolism of Long-Distance Transported Beef Cattle

Pre-slaughter long-distance transport resulted in a rapid depletion of muscle glycogen and led to a higher rate of dark, firm and dry (DFD) meat. Therefore, enhancing muscle glycogen reserves is critical for beef cattle prior to transportation. Creatine pyruvate (CrPyr) can provide simultaneous pyruvate and creatine and both are proven to promote the glycogen reserves. This study aimed to investigate the effects of transport treatment and dietary supplementation of rumen-protected (RP)-CrPyr on the meat quality, muscle energy metabolism, and hepatic gluconeogenesis of beef cattle. Twenty 18 month-old male Simmental crossbred cattle (659 ± 16 kg) were allotted 4 treatments based on a 2 × 2 factorial arrangement with two RP-CrPyr levels (140 g/d or 0 g/d) and two transport treatments (12 h or 5 min): ST_CrPyr0, ST_CrPyr140, LT_CrPyr0 and LT_CrPyr140. Three cattle per group were slaughtered after 30 days of feeding. The interaction of transport and RP-CrPyr had a significant effect on the muscle pH45 min, redness, glycogen content, GP, and AMP level (P < 0.05). Compared with short-distance transport, long-distance transport increased the muscle pH45 min value, redness, yellowness, drip loss, creatine level (P < 0.05), decreased muscle glycogen content, glycolytic potential (GP), and liver glucose amount (P < 0.05). Supplementation of RP-CrPyr decreased the activities of creatine kinase and lactate dehydrogenase in serum, muscle pH24 h value, redness, yellowness, lactate content, AMP level, and AMP/ATP (P < 0.05), increased the muscle glycogen content, GP, hexokinase activity, ATP and ADP levels, and ATP/ADP, liver pyruvate and glucose contents, activity of pyruvate carboxylase in the liver of cattle than those in the nonsupplemented treatments (P < 0.05). These results indicated that dietary RP-CrPyr supplementation might be favorable to improve meat quality and regulatory capacity of energy metabolism of beef cattle suffering long-distance transport followed with recovery treatment by increasing muscle glycogen storage, energy supply, and hepatic gluconeogenesis.

Regulation of Hepatic Lipid and Glucose Metabolism by INSP3R1.

With the rising epidemics of obesity and nonalcoholic fatty liver disease (NAFLD) and its downstream consequences including steatohepatitis, cirrhosis, and type 2 diabetes in the U.S. and worldwide, new therapeutic approaches are urgently needed to treat these devastating conditions. Glucagon, known for a century to be a glucose-raising hormone and clearly demonstrated to contribute to fasting and postprandial hyperglycemia in both type 1 and type 2 diabetes, represents an unlikely target to improve health in those with metabolic syndrome. However, recent work from our group and others' identifies an unexpected role for glucagon as a potential means of treating NAFLD, improving insulin sensitivity, and improving the lipid profile. We propose a unifying, calcium-dependent mechanism for glucagon's effects both to stimulate hepatic gluconeogenesis and to enhance hepatic mitochondrial oxidation: signaling through the inositol 1,4,5-trisphosphate receptor type 1 (INSP3R1), glucagon activates phospholipase C (PKC)/protein kinase A (PKA) signaling to enhance adipose triglyceride lipase (ATGL)-dependent intrahepatic lipolysis and, in turn, increase cytosolic gluconeogenesis by allosteric activation of pyruvate carboxylase. Simultaneously in the mitochondria, calcium transferred through mitochondria-associated membranes activates several dehydrogenases in the tricarboxylic acid cycle, correlated with an increase in mitochondrial energy expenditure and reduction in ectopic lipid. This model suggests that short-term, cyclic treatment with glucagon or other INSP3R1 antagonists could hold promise as a means to reset lipid homeostasis in patients with NAFLD.

The long noncoding RNA ADIPINT regulates human adipocyte metabolism via pyruvate carboxylase

The pleiotropic function of long noncoding RNAs is well recognized, but their direct role in governing metabolic homeostasis is less understood. Here, we describe a human adipocyte-specific lncRNA, ADIPINT, that regulates pyruvate carboxylase, a pivotal enzyme in energy metabolism. We developed an approach, Targeted RNA-protein identification using Orthogonal Organic Phase Separation, which identifies that ADIPINT binds to pyruvate carboxylase and validated the interaction with electron microscopy. ADIPINT knockdown alters the interactome and decreases the abundance and enzymatic activity of pyruvate carboxylase in the mitochondria. Reduced ADIPINT or pyruvate carboxylase expression lowers adipocyte lipid synthesis, breakdown, and lipid content. In human white adipose tissue, ADIPINT expression is increased in obesity and linked to fat cell size, adipose insulin resistance, and pyruvate carboxylase activity. Thus, we identify ADIPINT as a regulator of lipid metabolism in human white adipocytes, which at least in part is mediated through its interaction with pyruvate carboxylase.

Abstract #1184551: Hypertriglyceridemic pancreatitis in a rare case of type A pyruvate carboxylase deficiency

Pyruvate carboxylase (PC) deficiency is a spectrum of rare autosomal recessive disorders that can have significant metabolic consequences. Decreases of PC activity limits utilization of lactate and alanine for anaplerotic and synthetic fluxes. PC deficiency can lead to hypoglycemia, lactic acidosis, and hyperammonemia. Patients can develop central nervous system disease (via glial damage and demyelination leading to hydrocephalus), hepatotoxicity and insulinopenia. The latter can result in hypertriglyceridemia, though not a hallmark of PC deficiency.

Long noncoding RNA ZFP36L2-AS functions as a metabolic modulator to regulate muscle development

Skeletal muscle is the largest metabolic organ in the body, and its metabolic flexibility is essential for maintaining systemic energy homeostasis. Metabolic inflexibility in muscles is a dominant cause of various metabolic disorders, impeding muscle development. In our previous study, we found lncRNA ZFP36L2-AS (for “ZFP36L2-antisense transcript”) is specifically enriched in skeletal muscle. Here, we report that ZFP36L2-AS is upregulated during myogenic differentiation, and highly expressed in breast and leg muscle. In vitro, ZFP36L2-AS inhibits myoblast proliferation but promotes myoblast differentiation. In vivo, ZFP36L2-AS facilitates intramuscular fat deposition, as well as activates fast-twitch muscle phenotype and induces muscle atrophy. Mechanistically, ZFP36L2-AS interacts with acetyl-CoA carboxylase alpha (ACACA) and pyruvate carboxylase (PC) to induce ACACA dephosphorylation and damaged PC protein stability, thus modulating muscle metabolism. Meanwhile, ZFP36L2-AS can activate ACACA to reduce acetyl-CoA content, which enhances the inhibition of PC activity. Our findings present a novel model about the regulation of lncRNA on muscle metabolism.

Antibacterial Activity of the Essential Oil From Litsea cubeba Against Cutibacterium acnes and the Investigations of Its Potential Mechanism by Gas Chromatography-Mass Spectrometry Metabolomics.

Cutibacterium acnes (C. acnes) is an anaerobic Gram-positive bacterium generally considered as a human skin commensal, but is also involved in different infections, such as acne and surgical infections. Although there are a variety of treatments, the side effects and the problem of bacterial drug resistance still limit their clinical usage. In this study, we found that essential oil (EO) distilled from fresh mature Litsea cubeba possessed promising antibacterial activity against C. acnes. In order to elucidate its potential mechanism, bacteriostatic activity test, Live/Dead kit assay, scanning electron microscope (SEM), transmission electron microscope (TEM), and metabolomics were employed. In addition, the content of adenosine triphosphate (ATP) in bacterium and the activities of key enzymes involved in critical metabolic pathways were detected using a variety of biochemical assays. The results showed that EO exhibited significant antibacterial activity against C. acnes at a minimum inhibitory concentration (MIC) of 400 μg/mL and a minimum bactericidal concentration (MBC) of 800 μg/mL, and EO could destroy C. acnes morphology and inhibit its growth. Moreover, results from our study showed that EO had a significant effect on the C. acnes normal metabolism. In total, 86 metabolites were altered, and 34 metabolic pathways related to the carbohydrate metabolism, energy metabolism, amino acid metabolism, as well as cell wall and cell membrane synthesis were perturbed after EO administration. The synthesis of ATP in bacterial cells was also severely inhibited, and the activities of key enzymes of the glycolysis and Wood-Werkman cycle were significantly affected (Pyruvate Carboxylase, Malate Dehydrogenase and Pyruvate kinase activities were decreased, and Hexokinase was increased). Taken together, these results illustrated that the bacteriostatic effect of EO against C. acnes by breaking the bacterial cell morphology and perturbing cell metabolism, including inhibition of key enzyme activity and ATP synthesis. The results from our study may shed new light on the discovery of novel drugs with more robust efficacy.

A Short Isoform of Spermatogenic Enzyme GAPDHS Functions as a Metabolic Switch and Limits Metastasis in Melanoma.

This study characterizes metabolic changes during cancer metastasis and identifies GAPDHS as a novel regulator of these processes in melanoma cells.

Erianin suppresses proliferation and migration of cancer cells in a pyruvate carboxylase-dependent manner

Erianin is a natural small molecule dibenzyl compound extracted from Dendrobium officinale or Dendrobium chrysotoxum. Studies show erianin has many pharmacological functions such as antioxidant, antibacterial, antiviral, improving diabetic nephropathy, relaxing bronchial smooth muscle and anti-tumor. However, the erianin-mediated molecular mechanism is elusive, and the target protein of erianin is not clear yet. Here, we screened and identified that the target protein of erianin in human hepatoma HepG2 cells is human pyruvate carboxylase, and explored the anti-tumor signal pathway regulated by erianin in several cell lines. Firstly, the interaction between human pyruvate carboxylase and erianin was studied by bioinformatics and biochemical methods. Secondly, in vitro, erianin can specifically inhibit the activity of human pyruvate carboxylase, and the purified human pyruvate carboxylase can specifically bind to the activity probe of erianin. Thirdly, human pyruvate carboxylase is highly expressed in a variety of malignant tumors, and the inhibitory effect of erianin on tumor cells is positively correlated with the expression of human pyruvate carboxylase, and erianin can selectively inhibit the activity of pyruvate carboxylase. Finally, erianin can regulate the pyruvate carboxylase-mediated Wnt/ β- Catenin pathway. All of which provide important data for the further study of the anticancer mechanism of erianin, and lay a solid foundation for the further development and utilization of erianin.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Identification of Pyruvate Carboxylase as the Cellular Target of Natural Bibenzyls with Potent Anticancer Activity against Hepatocellular Carcinoma via Metabolic Reprogramming.

Cancer cell proliferation in some organs often depends on conversion of pyruvate to oxaloacetate via pyruvate carboxylase (PC) for replenishing the tricarboxylic acid cycle to support biomass production. In this study, PC was identified as the cellular target of erianin using the photoaffinity labeling-click chemistry-based probe strategy. Erianin potently inhibited the enzymatic activity of PC, which mediated the anticancer effect of erianin in human hepatocellular carcinoma (HCC). Erianin modulated cancer-related gene expression and induced changes in metabolic intermediates. Moreover, erianin promotes mitochondrial oxidative stress and inhibits glycolysis, leading to insufficient energy required for cell proliferation. Analysis of 14 natural analogs of erianin showed that some compounds exhibited potent inhibitory effects on PC. These results suggest that PC is a cellular target of erianin and reveal the unrecognized function of PC in HCC tumorigenesis; erianin along with its analogs warrants further development as a novel therapeutic strategy for the treatment of HCC.

Coordinated reprogramming of metabolism and cell function in adipocytes from proliferation to differentiation

Adipose tissue plays a major role in regulating lipid and energy homeostasis by storing excess nutrients, releasing energetic substrates through lipolysis, and regulating metabolism of other tissues and organs through endocrine and paracrine signaling. Adipocytes within fat tissues store excess nutrients through increased cell number (hyperplasia), increased cell size (hypertrophy), or both. The differentiation of pre-adipocytes into mature lipid-accumulating adipocytes requires a complex interaction of metabolic pathways that is still incompletely understood. Here, we applied parallel labeling experiments and 13C-metabolic flux analysis to quantify precise metabolic fluxes in proliferating and differentiated 3T3-L1 cells, a widely used model to study adipogenesis. We found that morphological and biomass composition changes in adipocytes were accompanied by significant shifts in metabolic fluxes, encompassing all major metabolic pathways. In contrast to proliferating cells, differentiated adipocytes 1) increased glucose uptake and redirected glucose utilization from lactate production to lipogenesis and energy generation; 2) increased pathway fluxes through glycolysis, oxidative pentose phosphate pathway and citric acid cycle; 3) reduced lactate secretion, resulting in increased ATP generation via oxidative phosphorylation; 4) rewired glutamine metabolism, from glutaminolysis to de novo glutamine synthesis; 5) increased cytosolic NADPH production, driven mostly by increased cytosolic malic enzyme flux; 6) increased production of monounsaturated C16:1; and 7) activated a mitochondrial pyruvate cycle through simultaneous activity of pyruvate carboxylase, malate dehydrogenase and malic enzyme. Taken together, these results quantitatively highlight the complex interplay between pathway fluxes and cell function in adipocytes, and suggest a functional role for metabolic reprogramming in adipose differentiation and lipogenesis.

Fibroblast pyruvate carboxylase is required for collagen production in the tumour microenvironment.

The aberrant production of collagen by fibroblasts is a hallmark of many solid tumors and can influence cancer progression. How the mesenchymal cells in the tumor microenvironment maintain their production of extracellular matrix proteins as the vascular delivery of glutamine and glucose becomes compromised remains unclear. Here we show that pyruvate carboxylase (PC)-mediated anaplerosis in tumor-associated fibroblasts contributes to tumor fibrosis and growth. Using cultured mesenchymal and cancer cells, as well as mouse allograft models, we provide evidence that extracellular lactate can be utilized by fibroblasts to maintain TCA cycle anaplerosis and non-essential amino acid biosynthesis through PC activity. Furthermore, we show that fibroblast PC is required for collagen production in the tumor microenvironment. These results establish TCA cycle anaplerosis as a determinant of extracellular matrix collagen production, and identify PC as potential target to inhibit tumor desmoplasia.

Effects of melatonin treatment on ethanol fermenation and ERF expression in kiwifruit cv. Bruno during postharvest

Kiwifruit (Actinidia deliciosa) cv. Bruno is easy to accumulate ethanol during postharvest, which seriously affects fruit quality. In this study, the effects of melatonin (MT) treatment on ethanol accumulation and AdERF genes relative expression in kiwifruit were investigated. The main results were as follows: MT treatment slowed down the fruit softening by inhibiting the rates of respiration and ethylene release, and significantly reduced the acetaldehyde and ethanol contents in kiwifruit during the later storage. Correspondingly, MT treatment significantly down-regulated the relative expressions of AdADH1, AdPDC1 and AdPDC2, and inhibited the activities of pyruvate carboxylase (PDC) and alcohol dehydrogenase (ADH) in kiwifruit during the later storage period. Also, MT treatment down-regulated the expressions of AdERF4, AdERF74, AdERF75 and Achn226291 during the later storage, while the relative expressions of AdERF5, AdERF6, AdERF15, Achn164421 and Achn330401 were not significantly changed. These results indicated that MT treatment could affect the ethanol metabolism in kiwifruit by regulating the expressions of ADH1, PDC1 and PDC2 as well as the activities of ADH and PDC, thereby contributing to the quality maintenance and the shelf-life extension of kiwifruit. AdERF4, AdERF74, AdERF75 and Achn226291 might be implicated in the process. This study provided a theoretical basis for the MT application in maintaining fruit quality associated with control of alcoholic off-flavor via regulation of ethanol fermentation in kiwifruit during postharvest.

Semi-rational evolution of pyruvate carboxylase from Rhizopus oryzae for elevated fumaric acid synthesis in Saccharomyces cerevisiae

Dicarboxylic acids are widely used in food, pharmaceutical, and chemical industries. Pyruvate carboxylase (PYC) plays a pivotal role in the production of dicarboxylic acids in microbial fermentation process. Our previous work showed that heterologous expression of pyruvate carboxylase (RoPYC) from Rhizopus oryzae resulted in an increase in fumaric acid titer to 226.0 ± 2.2 mg/L from 194.0 ± 4.0 mg/L in the S. cerevisiae pdc1adh1fum1 strain. However, PYC still remained the metabolic step limiting the production of target carboxylic acids. In this study, semi-rational evolution of pyruvate carboxylase by site-saturation mutagenesis combined with codon optimization was conducted to further improve fumaric acid synthesis. We demonstrated that each of three mutations (N315F, R485P and N1078F) or codon optimization of RoPYC significantly increased the production of fumaric acid. A maximal titer of 465.5 ± 6.5 mg/L was achieved in flasks by the strain expressing codon-optimized RoPYC mutant (R485P). Enzyme assays of these mutants showed higher PYC activities, while homology modeling indicated that the increased PYC activities could be attributed to the modulation of the allosteric domain and the biotin carboxylation domain. In addition, both calcium ion and carbon dioxide displayed positive effects on the fumaric acid production by this mutant. Overall, the strategy described here demonstrated an effective way for elevating PYC activity and further enhance the synthesis of dicarboxylic acids.