Overexpression Of Pyruvate Carboxylase Research Articles

Anemoside B4, a new pyruvate carboxylase inhibitor, alleviates colitis by reprogramming macrophage function.

Colitis is a global disease usually accompanied by intestinal epithelial damage and intestinal inflammation, and an increasing number of studies have found natural products to be highly effective in treating colitis. Anemoside B4 (AB4), an abundant saponin isolated from Pulsatilla chinensis (Bunge), whichwas found to have strong anti-inflammatory activity. However, the exact molecular mechanisms and direct targets of AB4 in the treatment of colitis remain to be discovered. The anti-inflammatory activities of AB4 were verified in LPS-induced cell models and 2, 4, 6-trinitrobenzene sulfonic (TNBS) or dextran sulfate sodium (DSS)-induced colitis mice and rat models. The molecular target of AB4 was identified by affinity chromatography analysis using chemical probes derived from AB4. Experiments including proteomics, molecular docking, biotin pull-down, surface plasmon resonance (SPR), and cellular thermal shift assay (CETSA) were used to confirm the binding of AB4 to itsmolecular target. Overexpression of pyruvate carboxylase (PC) and PC agonist were used to study the effects of PC on the anti-inflammatory and metabolic regulation of AB4 in vitro and in vivo. AB4 not only significantly inhibited LPS-induced NF-κB activation and increased ROS levels in THP-1 cells, but also suppressed TNBS/DSS-induced colonic inflammation in mice and rats. The molecular target of AB4 was identified as PC, a key enzyme related to fatty acid, amino acid and tricarboxylic acid (TCA) cycle. We next demonstrated that AB4 specifically bound to the His879 site of PC and altered the protein's spatial conformation, thereby affecting the enzymatic activity of PC. LPS activated NF-κB pathway and increased PC activity, which caused metabolic reprogramming, while AB4 reversed this phenomenon by inhibiting the PC activity. In vivo studies showed that diisopropylamine dichloroacetate (DADA),a PC agonist, eliminated the therapeutic effects of AB4 by changing the metabolic rearrangement of intestinal tissues in colitis mice. We identified PC as a direct cellular target of AB4 in the modulation of inflammation, especially colitis. Moreover, PC/pyruvate metabolism/NF-κB is crucial for LPS-driven inflammation andoxidative stress. These findings shed more light on thepossibilities ofPC as a potential new target for treating colitis.

Iterative optimization of exogenous genes and redirection of metabolic flux for enhanced itaconate biosynthesis in engineered Escherichia coli

BackgroundItaconate or itaconic acid (IA), a sustainable and valuable platform chemical, has garnered significant attention due to its potential as an alternative petroleum-derived compound. In the past, Aspergillus terreus offered a high titer of IA, but it still faced the challenges of low productivity. To overcome the problem, a fast-growing Escherichia coli BW25113 was involved consolidating the itaconate production pathway, eliminating byproduct-forming pathways, and enhancing intermediate levels to facilitate large-scale production. MethodE. coli was engineered through the deletion of isocitrate decarboxylase (Icd) and the overexpression of pyruvate carboxylase (CgPyc), cis-aconitate decarboxylase (AtCadA) and aconitate hydratase (ENAcnA) to facilitate the IA biosynthesis pathway. A B0015 terminator was encoded behind AtCadA to improve transcription efficiency as well as poxB gene was deleted to accelerate carbon influx towards the tricarboxylic acid cycle instead of forming the byproduct acetate. A semi-continuous batch fermentation approach was employed for large-scale IA biosynthesis to address ATP insufficiency in long-termed fermentation. Significant findingThe engineered strain AB15C/Δicd improved IA titer by 20 % compared to the control strain. By deleting the acetate forming pathway under acidic condition, AB15C/ΔicdΔpoxB increased itaconate production. Overexpression of ENAcnA in AB15CEN*/ΔicdΔpoxB strain resulted in 13.8 g/L of IA. Finally, a semi-continuous batch fermentation was implemented, leading to an IA accumulation of 35.5 g/L after 4 batches.

Abstract 1267: Suppression of pyruvate carboxylase drives PDL1 expression in mouse models of TNBC

Abstract Background: Programmed death-ligand 1 (PDL1) expression on tumor and other cells dampens T cell activation and promotes exhaustion. Pyruvate carboxylase (PC) converts pyruvate to oxaloacetate which in T cells and tumor associated macrophages supports antitumor immunity. PC is required for lung metastasis in several models of triple negative breast cancer (TNBC); however the role of PC expression in primary tumor growth is less well understood. Suppression of PC in tumor associated macrophages and T cells blunts antitumor immunity. We have shown that suppression of PC in tumor cells is sufficient to promote tumor progression and immune evasion. Here we investigated in murine models of TNBC whether and how PC suppression might alter the expression of PDL1. Methods: PC was stably suppressed in M-Wnt, metM-Wntlung and E0771 cells using shRNA, and transiently suppressed using siRNA. C57BL6NCrl mice were orthotopically injected with shPC and scram M-Wnt (n=8/group) or E0771 (n=5/group) cells. Transcriptomic analysis of E0771 cells was performed using Clariom D microarray. PC and PDL1 expression was determined in M-Wnt and metM-Wntlung cells by qPCR. Cell signaling was assayed by western blot using pERK and pAKT antibodies. Statistical analysis was performed using two-sided students t-test, with Welch’s correction where variance was not equal. Results: Suppression of PC in M-Wnt and E0771 cells promotes tumor growth. In vitro, suppression of PC promoted PDL1 expression in E0771 cells. We confirmed similar effects in M-Wnt and metM-Wntlung cells stably expressing shRNA directed against PC using qPCR. Finally we confirmed that transient transfection of siRNA directed against PC similarly induced PDL1 expression. Basal AKT and ERK signaling was induced by stable suppression of PC in M-Wnt cells. Conclusions: PC suppression in several murine cell models of TNBC promotes PDL1 expression and reveals an underexplored interaction between tumor cell anaplerosis and immune evasion. Through suppression of PC we have identified a reciprocal relationship between PC and PDL1 expression, with a concomitant induction of both AKT and ERK signaling. A better understanding of the mechanisms through which PC-related metabolic reprogramming modulates antitumor immunity will identify new targets for inhibiting TNBC progression. Future studies will address whether targeted restoration of anaplerosis or overexpression of PC can suppress PDL1 and promote antitumor immunity. Citation Format: Numair Attaar, Alexander J. Pfeil, Eylem K. Cotul, Dorothy Teegarden, Michael K. Wendt, Michael F. Coleman, Stephen D. Hursting. Suppression of pyruvate carboxylase drives PDL1 expression in mouse models of TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1267.

Metabolic Engineering of Trichoderma reesei for l-Malic Acid Production.

l-Malic acid has various applications in the chemical and food industries. The filamentous fungus Trichoderma reesei is known to be an efficient enzyme producer. Here, through metabolic engineering, T. reesei was constructed for the first time as an excellent cell factory for l-malic acid production. The heterologous overexpression of genes encoding the C4-dicarboxylate transporter from Aspergillus oryzae and Schizosaccharomyces pombe initiated l-malic acid production. The overexpression of pyruvate carboxylase from A. oryzae in the reductive tricarboxylic acid pathway further increased both the titer and yield of l-malic acid, resulting in the highest titer reported in a shake-flask culture. Furthermore, the deletion of malate thiokinase blocked l-malic acid degradation. Finally, the engineered T. reesei strain produced 220.5 g/L of l-malic acid in a 5 L fed-batch culture (productivity of 1.15 g/L/h). A T. reesei cell factory was created for the efficient production of l-malic acid.

Overexpression of Pyruvate Carboxylase Is Correlated With Colorectal Cancer Progression and Supports Growth of Invasive Colon Cancer HT-29 Cell Line.

Pyruvate carboxylase (PC) is a major anaplerotic enzyme for generating oxaloacetate for the TCA cycle and also a key enzyme in gluconeogenesis, de novo fatty acid and amino acid synthesis in normal cells. Recent studies have identified PC overexpression in different cancers, such as breast and lung. However, the involvement of PC in colorectal cancer (CRC) is unclear. Our purpose was to investigate the PC expression levels and its correlations with potentially relevant clinical-pathological parameters in CRC. PC expression levels in tissues from 60 Thai CRC patients were investigated by immunohistochemistry while a clonogenic assay was performed for determining cell growth of HT-29 cells with PC knockdown. Our results showed for the first time that high PC expression levels were significantly correlated with late stage of the cancer, perineural invasion and lymph node metastasis. The overexpression of PC was also significantly associated with poor overall and disease-free survival times of CRC patients. In addition, suppression of cancer cell growth was found in PC-deficient cell lines using CRISPR-Cas9. The overexpression levels of PC were correlated with CRC progression and survival times. Therefore, PC might serve as a potential clinical prognostic marker for colorectal cancer.

C-Myc directly targets an over-expression of pyruvate carboxylase in highly invasive breast cancer

Here we showed that the c-Myc oncogene is responsible for overexpression of pyruvate carboxylase (PC) in highly invasive MDA-MB-231 cells. Pharmacological inhibition of c-Myc activity with 10074-G5 compound, resulted in a marked reduction of PC mRNA and protein, concomitant with reduced cell growth, migration and invasion. This growth inhibition but not migration and invasion can be partly restored by overexpression of PC, indicating that PC is a c-Myc-regulated pro-proliferating enzyme. Analysis of chromatin immunoprecipitation sequencing of c-Myc bound promoters revealed that c-Myc binds to two canonical c-Myc binding sites, locating at nucleotides −417 to −407 and −301 to −291 in the P2 promoter of human PC gene. Mutation of either c-Myc binding site in the P2 promoter-luciferase construct resulted in 50–60% decrease in luciferase activity while double mutation of c-Myc binding sites further decreased the luciferase activity in MDA-MB-231 cells. Overexpression of c-Myc in HEK293T cells that have no endogenous c-Myc resulted in 250-fold increase in luciferase activity. Mutation of either E-boxes lowered luciferase activity by 50% and 25%, respectively while double mutation of both sites abolished the c-Myc transactivation response. An electrophoretic mobility shift assay using nuclear proteins from MDA-MB-231 confirmed binding of c-Myc to both c-Myc binding sites in the P2 promoter. Bioinformatic analysis of publicly available transcriptomes from the cancer genome atlas (TCGA) dataset revealed an association between expression of c-Myc and PC in primary breast, as well as in lung and colon cancer tissues, suggesting that overexpression of PC is deregulated by c-Myc in these cancers.

Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production

Ustilago trichophora RK089 has been found recently as a good natural malic acid producer from glycerol. This strain has previously undergone adaptive laboratory evolution for enhanced substrate uptake rate resulting in the strain U. trichophora TZ1. Medium optimization and investigation of process parameters enabled titers and rates that are able to compete with those of organisms overexpressing major parts of the underlying metabolic pathways. Metabolic engineering can likely further increase the efficiency of malate production by this organism, provided that basic genetic tools and methods can be established for this rarely used and relatively obscure species.Here we investigate and adapt existing molecular tools from U. maydis for use in U. trichophora. Selection markers from U. maydis that confer carboxin, hygromycin, nourseothricin, and phleomycin resistance are applicable in U. trichophora. A plasmid was constructed containing the ip-locus of U. trichophora RK089, resulting in site-specific integration into the genome. Using this plasmid, overexpression of pyruvate carboxylase, two malate dehydrogenases (mdh1, mdh2), and two malate transporters (ssu1, ssu2) was possible in U. trichophora TZ1 under control of the strong Petef promoter. Overexpression of mdh1, mdh2, ssu1, and ssu2 increased the product (malate) to substrate (glycerol) yield by up to 54% in shake flasks reaching a titer of up to 120gL−1. In bioreactor cultivations of U. trichophora TZ1 Petefssu2 and U. trichophora TZ1 Petefmdh2 a drastically lowered biomass formation and glycerol uptake rate resulted in 29% (Ssu1) and 38% (Mdh2) higher specific production rates and 38% (Ssu1) and 46% (Mdh2) increased yields compared to the reference strain U. trichophora TZ1. Investigation of the product spectrum resulted in an 87% closed carbon balance with 134gL−1 malate and biomass (73gL−1), succinate (20gL−1), CO2 (17gL−1), and α-ketoglutarate (8gL−1) as main by-products.These results open up a wide range of possibilities for further optimization, especially combinatorial metabolic engineering to increase the flux from pyruvate to malic acid and to reduce by-product formation.

Improving Process Yield in Succinic Acid Production by Cell Recycling of Recombinant Corynebacterium glutamicum

Aerobically cultivated cells of Corynebacterium glutamicum produce mixed organic acids, including succinic acid (SA), from glucose when the cells are transferred to oxygen-deprived conditions. Genetic modification, including inactivation of lactate dehydrogenase and overexpression of pyruvate carboxylase, allows this microbe to be an efficient SA producer under the conditions of oxygen deprivation. High productivity and high titers can be achieved in the production process by using the genetically engineered strain of C. glutamicum under the given conditions. However, glucose consumption for cell preparation decreases process yield (defined as the quantity of SA produced divided by the total quantity of glucose used in cell preparation and SA production). In this study, we investigated cell recycle fed-batch fermentation for SA production to improve the process yield by reducing the effect of glucose consumption for cell preparation on the process yield. A genetically stable and markerless strain, harboring nine genomic copies of the pyruvate carboxylase gene, was newly constructed and used for cell recycle fermentation. During 26 reaction cycles, only 0.7% decrease in specific productivity per reaction was observed. Overall, the process yield was improved by 79% compared to that in a single fed-batch reaction without cell recycling.

Pyruvate Carboxylase Is Up-Regulated in Breast Cancer and Essential to Support Growth and Invasion of MDA-MB-231 Cells.

Pyruvate carboxylase (PC) is an anaplerotic enzyme that catalyzes the carboxylation of pyruvate to oxaloacetate, which is crucial for replenishing tricarboxylic acid cycle intermediates when they are used for biosynthetic purposes. We examined the expression of PC by immunohistochemistry of paraffin-embedded breast tissue sections of 57 breast cancer patients with different stages of cancer progression. PC was expressed in the cancerous areas of breast tissue at higher levels than in the non-cancerous areas. We also found statistical association between the levels of PC expression and tumor size and tumor stage (P < 0.05). The involvement of PC with these two parameters was further studied in four breast cancer cell lines with different metastatic potentials; i.e., MCF-7, SKBR3 (low metastasis), MDA-MB-435 (moderate metastasis) and MDA-MB-231 (high metastasis). The abundance of both PC mRNA and protein in MDA-MB-231 and MDA-MB-435 cells was 2-3-fold higher than that in MCF-7 and SKBR3 cells. siRNA-mediated knockdown of PC expression in MDA-MB-231 and MDA-MB-435 cells resulted in a 50% reduction of cell proliferation, migration and in vitro invasion ability, under both glutamine-dependent and glutamine-depleted conditions. Overexpression of PC in MCF-7 cells resulted in a 2-fold increase in their proliferation rate, migration and invasion abilities. Taken together the above results suggest that anaplerosis via PC is important for breast cancer cells to support their growth and motility.

Metabolic engineering of Torulopsis glabrata for malate production

The yeast Torulopsis glabrata CCTCC M202019, which is used for industrial pyruvate production, was chosen to explore the suitability of engineering this multi-vitamin auxotrophic yeast for increased malate production. Various metabolic engineering strategies were used to manipulate carbon flux from pyruvate to malate: (i) overexpression of pyruvate carboxylase and malate dehydrogenase; (ii) identification of the bottleneck in malate production by model iNX804; (iii) simultaneous overexpression of genes RoPYC, RoMDH and SpMAE1. Using these strategies, 8.5gL–1 malate was accumulated in the engineered strain T.G-PMS, which was about 10-fold greater than that of the control strain T.G-26. The results presented here suggest that T. glabrata CCTCC M202019 is a promising candidate for industrial malate production.

Efficient Succinic Acid Production from Glucose through Overexpression of Pyruvate Carboxylase in an Escherichia coli Alcohol Dehydrogenase and Lactate Dehydrogenase Mutant

An adhE, ldhA double mutant Escherichia coli strain, SBS110MG, has been constructed to produce succinic acid in the presence of heterologous pyruvate carboxylase (PYC). The strategic design aims at diverting maximum quantities of NADH for succinate synthesis by inactivation of NADH competing pathways to increase succinate yield and productivity. Additionally an operational PFL enzyme allows formation of acetyl-CoA for biosynthesis and formate as a potential source of reducing equivalents. Furthermore, PYC diverts pyruvate toward OAA to favor succinate generation. SBS110MG harboring plasmid pHL413, which encodes the heterologous pyruvate carboxylase from Lactococcus lactis, produced 15.6 g/L (132 mM) of succinate from 18.7 g/L (104 mM) of glucose after 24 h of culture in an atmosphere of CO(2) yielding 1.3 mol of succinate per mole of glucose. This molar yield exceeded the maximum theoretical yield of succinate that can be achieved from glucose (1 mol/mol) under anaerobic conditions in terms of NADH balance. The current work further explores the importance of the presence of formate as a source of reducing equivalents in SBS110MG(pHL413). Inactivation of the native formate dehydrogenase pathway (FDH) in this strain significantly reduced succinate yield, suggesting that reducing power was lost in the form of formate. Additionally we investigated the effect of ptsG inactivation in SBS110MG(pHL413) to evaluate the possibility of a further increase in succinate yield. Elimination of the ptsG system increased the succinate yield to 1.4 mol/mol at the expense of a reduction in glucose consumption of 33%. In the presence of PYC and an efficient conversion of glucose to products, the ptsG mutation is not indispensable since PEP converted to pyruvate as a result of glucose phosphorylation by the glucose specific PTS permease EIICB(glu) can be rediverted toward OAA favoring succinate production.

The role of pyruvate carboxylase in insulin secretion and proliferation in rat pancreatic beta cells.

Pyruvate carboxylase (PC) or pyruvate dehydrogenase (PDH) is required to transfer carbons from pyruvate into the Krebs cycle. PC activity is preserved in the islets of obese animals, but it is reduced in the islets of animal models of type 2 diabetes, suggesting that PC is important in beta cell adaptation to insulin resistance and that PC reduction may lead to beta cell failure. To confirm the significance of PC, we first lowered activity using Pc (now known as Pcx) small interfering RNA (siRNA) in INS-1 cells and in dispersed rat islet cells. Second, we overexpressed PC in INS-1 cells, and third, we inhibited PDH by overexpressing the gene encoding pyruvate dehydrogenase kinase 4 (Pdk4) in INS-1 cells. Treatment of INS-1 cells or dispersed rat islet cells with Pc siRNA resulted in a significant reduction in insulin secretion in both cell types and reduced proliferation in INS-1 cells. This treatment also reduced the content of oxaloacetate, malate and ATP, as well as the NADPH:NADP(+) ratio and activity of the pyruvate-malate shuttle. Overexpression of PC in INS-1 cells led to an elevation of insulin secretion and cell proliferation, whereas inhibition of PDH activity by overexpressing Pdk4 in INS-1 cells did not reduce insulin secretion. Our findings indicate that the PC pathway in beta cells might play a key role in pyruvate metabolism, insulin secretion and cell proliferation.

Effect of aniA (carbon flux regulator) and PhaC (poly-beta-hydroxybutyrate synthase) mutations on pyruvate metabolism in Rhizobium etli.

The Rhizobium etli poly-beta-hydroxybutyrate synthase (PhaC) mutant SAM100 grows poorly with pyruvate as the carbon source. The inactivation of aniA, encoding a global carbon flux regulator, in SAM100 restores growth of the resulting double mutant (VEM58) on pyruvate. Pyruvate carboxylase (PYC) activity, pyc gene transcription, and holoenzyme content, which were low in SAM100, were restored in strain VEM58. The genetically engineered overexpression of PYC in SAM100 also allowed its growth on pyruvate. The possible relation between AniA, pyc transcription, and reduced-nucleotide levels is discussed.