PFK Expression Research Articles

Two Strains of Gibel Carp (Carassius gibelio) Exhibit Diverse Responses to Carbohydrates in a Low-Lipid Diet

An 8-week feeding trial was conducted to investigate carbohydrate use in a low-lipid diet between different strains of gibel carp (Carassius gibelio). The wild-type Dongting strain and artificially selected CASIII strain were fed three levels of carbohydrate: 15%, 30%, and 45%, in a low-lipid formulation (4%). The results showed that low-lipid supplementation with high carbohydrate seems to improve the feeding rate (FR) and specific growth rate (SGR), while it did not result in hyperglycaemia in gibel carp. Transcriptional regulation was slightly affected by carbohydrate levels; only hepatic pfk expression was more increased in the 30% and 45% carbohydrate diets than in the 15% carbohydrate diet ( p < 0.05 ). Regarding the strains, CASIII obtained higher growth performance by having higher FR and SGR than Dongting ( p < 0.05 ). Relatively higher glycaemia was found in CASIII, and it was coupled with increased plasma triglycerides and insulin ( p > 0.05 ). Enhanced gene expression of pk involved in glycolysis and decreased mRNA levels of pepck in gluconeogenesis were also observed in CASIII ( p < 0.05 ). There were strong strain ∗ diet interactions of gene expression involved in lipogenesis, and Dongting had higher mRNA levels of srebp-1, acly, and fas when fed carbohydrate at 45% ( p < 0.05 ). In conclusion, the tolerant level of carbohydrates in a low-lipid diet could be up to 45% for gibel carp by an improvement of growth and noninducement of postprandial plasma glucose. CASIII had a better ability to use dietary carbohydrates for growth with active glycolysis and restrained gluconeogenesis, and Dongting had more potential in lipid metabolism in response to carbohydrate intake.

A novel microRNA and its pfk target control growth length in the freshwater shrimp Neocaridina heteropoda.

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression and play roles in a wide range of physiological processes, including ontogenesis. Herein, we discovered a novel miRNA, novel miR-26, which inhibits translation of the phosphofructokinase (PFK) gene by targeting the 3' untranslated region (UTR) of pfk directly, thereby inhibiting molting and body length growth of the freshwater shrimp Neocaridina heteropoda Lowering expression of pfk by RNA interference (RNAi) led to a longer ecdysis cycle and smaller individuals. This phenotype was mirrored in shrimps injected with novel miR-26 agomirs, but the opposite phenotype occurred in shrimps injected with novel miR-26 antagomirs (i.e. the ecdysis cycle was shortened and body length was increased). After injection of 20-hydroxyecdysone (ecdysone 20E), expression of the novel miR-26 was decreased, while expression of pfk was up-regulated, and the fructose-1,6-diphosphate metabolite of PFK accumulated correspondingly. Furthermore, expression of eIF2 (eukaryotic initiation factor 2) increased under stimulation with fructose-1,6-diphosphate, suggesting that protein synthesis was stimulated during this period. Taken together, our results suggest that the novel miR-26 regulates expression of pfk and thereby mediates the molting and growth of N. heteropoda.

Expression of Phosphofructokinase Is Not Sufficient to Enable Embden-Meyerhof-Parnas Glycolysis in Zymomonas mobilis ZM4.

Zymomonas mobilis is a bacterium that produces ethanol from glucose at up to 97% of theoretical efficiency on a carbon basis. One factor contributing to the high efficiency of ethanol production is that Z. mobilis has a low biomass yield. The low biomass yield may be caused partly by the low ATP yield of the Entner-Doudoroff (ED) glycolytic pathway used by Z. mobilis, which produces only one ATP per glucose consumed. To test the hypothesis that ATP yield limits biomass yield in Z. mobilis, we attempted to introduce the Embden-Meyerhof-Parnas (EMP) glycolytic pathway (with double the ATP yield) by expressing phosphofructokinase (Pfk I) from Escherichia coli. Expression of Pfk I caused growth inhibition and resulted in accumulation of mutations in the pfkA gene. Co-expression of additional EMP enzymes, fructose bisphosphate aldolase (Fba) and triose phosphate isomerase (Tpi), with Pfk I did not enable EMP flux, and resulted in production of glycerol as a side product. Further analysis indicated that heterologous reactions may have operated in the reverse direction because of native metabolite concentrations. This study reveals how the metabolomic context of a chassis organism influences the range of pathways that can be added by heterologous expression.

Antimicrobial Mechanism of Oleanolic and Ursolic Acids on Streptococcus mutans UA159.

Triterpenoid saponin derivatives oleanolic acid (OA) and ursolic acid (UA), but not betulinic acid (BA), were previously found to have strong antimicrobial activity against Streptococcus mutans. OA and UA inhibited the transcription of genes related to peptidoglycan biosynthesis, thereby preventing bacterial growth. However, it is not clear whether this is the only pathway involved in the antimicrobial activity of these compounds against S. mutans. Therefore, we used quantitative real-time PCR (qPCR) and microarray analyses to examine the expression of genes related to essential metabolic pathways in S. mutans UA159 following incubation with OA, UA, or BA. An oligonucleotide array consisting of 5363 probes was designed to survey 1928 of the 1963 genes in the genome of S. mutans UA159. Genes that showed>2-fold changes in expression in response to the treatment conditions were annotated, and selected target genes involved in central metabolism were analyzed by qPCR. Microarray analysis confirmed that the gene expression patterns of the OA- and UA-treated cells differed from that of the BA-treated culture, indicating differences in the antimicrobial mechanism. In particular, the expression of pfk and pykF, coding for glycolysis regulatory proteins phosphofructokinase and pyruvate kinase, respectively, were significantly decreased in the OA and UA groups (P<0.05), as were genes involved in fatty acid and amino acid synthesis. In addition, the microarray analysis confirmed previous qPCR results showing that peptidoglycan synthesis is down-regulated in the OA- and UA-treated groups. OA and UA also appear to decrease the generation of organic acids by S. mutans UA159, which would have an anticaries effect. Overall, these findings suggest that OA and UA affect multiple genes involved in the central metabolism of S. mutans, with inhibition of glycolysis, fatty acid synthesis, amino acid synthesis, and peptidoglycan synthesis, all contributing to their antimicrobial activity.

The progression from a lower to a higher invasive stage of bladder cancer is associated with severe alterations in glucose and pyruvate metabolism

Cancer cells present a particular metabolic behavior. We hypothesized that the progression of bladder cancer could be accompanied by changes in cells glycolytic profile. We studied two human bladder cancer cells, RT4 and TCCSUP, in which the latter represents a more invasive stage. The levels of glucose, pyruvate, alanine and lactate in the extracellular media were measured by Proton Nuclear Magnetic Resonance. The protein expression levels of glucose transporters 1 (GLUT1) and 3 (GLUT3), monocarboxylate transporter 4 (MCT4), phosphofructokinase-1 (PFK1), glutamic-pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) were determined. Our data showed that glucose consumption and GLUT3 levels were similar in both cell lines, but TCCSUP cells displayed lower levels of GLUT1 and PFK expression. An increase in pyruvate consumption, concordant with the higher levels of lactate and alanine production, was also detected in TCCSUP cells. Moreover, TCCSUP cells presented lower protein expression levels of GPT and LDH. These results illustrate that bladder cancer progression is associated with alterations in cells glycolytic profile, namely the switch from glucose to pyruvate consumption in the more aggressive stage. This may be useful to develop new therapies and to identify biomarkers for cancer progression.

Modular pathway engineering of Bacillus subtilis for improved N-acetylglucosamine production

In previous work, we constructed a recombinant Bacillus subtilis strain for microbial production of N-acetylglucosamine (GlcNAc), which has applications in nutraceuticals and pharmaceuticals. In this work, we improve GlcNAc production through modular engineering of B. subtilis. Specifically, the GlcNAc synthesis-related metabolic network in B. subtilis was divided into three modules—GlcNAc synthesis, glycolysis, and peptidoglycan synthesis. First, two-promoter systems with different promoter types and strengths were used for combinatorial assembly of expression cassettes of glmS (encoding GlcN-6-phosphate synthase) and GNA1 (encoding GlcNAc-6-phosphate N-acetyltransferase) at transcriptional levels in the GlcNAc synthesis module, resulting in a 32.4% increase in GlcNAc titer (from 1.85g/L to 2.45g/L) in shake flasks. In addition, lactate and acetate synthesis were blocked by knockout of ldh (encoding lactate dehydrogenase) and pta (encoding phosphotransacetylase), leading to a 44.9% increase in GlcNAc production (from 2.45g/L to 3.55g/L) in shake flasks. Then, various strengths of the glycolysis and peptidoglycan synthesis modules were constructed by repressing the expression of pfk (encoding 6-phosphofructokinase) and glmM (encoding phosphoglucosamine mutase) via the expression of various combinations of synthetic small regulatory RNAs and Hfq protein. Next, GlcNAc, glycolysis, and peptidoglycan synthesis modules with various strengths were assembled and optimized via a module engineering approach, and the GlcNAc titer was improved to 8.30g/L from 3.55g/L in shake flasks. Finally, the GlcNAc titer was further increased to 31.65g/L, which was 3.8-fold that in the shake flask, in a 3-L fed-batch bioreactor. This work significantly enhanced GlcNAc production through modular pathway engineering of B. subtilis, and the engineering strategies used herein may be useful for the construction of versatile B. subtilis cell factories for the production of other industrially important chemicals.

Expression of key substrate cycle enzymes in rat spermatogenic cells: Fructose 1,6 bisphosphatase and 6 phosphofructose 1‐kinase

A substrate cycle composed of phosphofructo 1-kinase I (PFK) and fructose 1,6 bisphosphatase I (FBPase) has been proposed in rat spermatids. This substrate cycle can explain the ability of glucose to induce a decrease in intracellular ATP, a phenomenon that was related to regulation of [Ca(2+)]i in these cells. In spite of the importance of this metabolic cycle, the expression and activities of the enzymes that compose such cycle have not been systematically studied in spermatogenic cells. Here, we show that PFK and FBPase activities were present in pachytene spermatocytes and round spermatids extracts. Expression of PFK at the mRNA and protein levels showed a relatively similar expression in spermatogenic cells, but a stronger expression in Sertoli cells. Instead, expression of FBPase at the mRNA and protein levels was stronger in round and elongating spermatids as compared to other spermatogenic cells. A similar pattern was observed when evidencing FBPase activity by a NADPH-nitroblue tetrazolium-linked cytochemical assay in isolated pachytene spermatocytes and round spermatids. Rat spermatids also showed the ability to convert lactate to fructose- and glucose-6-P, indicating that both glycolytic and gluconeogenic fluxes are present in these cells. Our results indicate that a coordinated expression of key substrate cycle enzymes, at the level of PFK/FBPase, appear in the last stages of spermatogenic cell differentiation, suggesting that the co-regulation of these enzymes are required for the ability of these cells to respond to glucose and induce metabolic and Ca(2+) signals that can be important for sperm development and function.

Construction of an inducible, pfkA and pfkB deficient strain of Escherichia coli for the expression and purification of phosphofructokinase from bacterial sources

As the key obligatory step in the glycolytic pathway, the regulation of phosphofructokinase (PFK-1) has been the focus of study of several laboratories. While standard cloning procedures have opened the door to study PFK from a vast array of sources, a good pfk knockout Escherichia coli strain has not previously been developed. Many laboratories rely on DF1020 or similar derivatives for PFK expression. Unfortunately, DF1020 grows poorly and does not have an inherent means for controlling expression of genes from plasmids. More importantly, however, DF1020 has a tendency to grow on minimal media when glucose is used as the sole carbon source. In this study, a new E. coli PFK expression strain lacking both PFK-1 and PFK-2 has been engineered using λ-red mediated chromosomal deletion. The resulting strain has been designated RL257. In addition to having both pfkA and pfkB deleted, RL257 contains the lacI q allele, which allows for inducible expression when coupled with an expression vector containing either the lac or tac promoter.

Control of the Murine Phosphofructokinase-A Gene during Muscle Differentiation

The muscle-specific isoform of phosphofructokinase (PFK-A) is induced during muscle development. To understand expression of PFK at the molecular level, transcription of the mouse PFK-A gene was examined during C2 myoblast differentiation to myotubes. PFK-A gene transcription increased 5-7-fold during differentiation in vitro. To identify cis-acting elements which direct muscle-specific transcription of the PFK-A gene, its 5'-flanking region and first exon were cloned and characterized. S1 nuclease protection and primer extension assays showed four sites of transcription initiation at 106, 105, 88, and 87 bp upstream of the translation initiation codon. Stable transfection of fusion genes linking -1900 to +99 of PFK-A 5'-flanking sequence to chloramphenicol acetyltransferase coding sequences into myogenic C2 cells did not confer muscle-specific expression. However, larger fragments of PFK-A 5'-flanking region (-5800 to +99) showed muscle-specific expression by transient transfection assay. The sequences directing muscle-specific transcription were further defined by linking various PFK-A upstream fragments to the luciferase gene under the control of the PFK-A proximal promoter, -335 to +99 bp. We found DNA sequence responsible for muscle-specific expression of the PFK-A gene between -4800 and -3900 bp.

Molecular genetics of phosphofructokinase in the yeast Kluyveromyces lactis

We have undertaken a study of phosphofructokinase (PFK; E.C. 2.7.1.11) in the yeast Kluyveromyces lactis. Like other eukaryotic PFKs, the K. lactis enzyme is activated by the allosteric effectors AMP and fructose-2,6-bisphosphate. PFK activity is induced in cells grown on glucose as compared to ethanol-grown cells, in contrast to the constitutive expression of PFK in Saccharomyces cerevisiae. We show here that phosphofructokinase of the yeast K. lactis is composed of two non-identical types of subunits, encoded by the genes KIPFK1 and KIPFK2. We have cloned and sequenced both genes. KIPFK1 and KIPFK2 encode the alpha- and the beta-PFK subunits with deduced molecular weights of 109.336 Da and 104.074 Da, respectively. Sequence analysis indicates that the genes evolved from a double duplication event. Null mutants in either of the genes lack detectable PFK activity in vitro and the respective subunits cannot be detected on Western blots. In contrast to the situation in S. cerevisiae, Klpfk1 Klpfk2 double mutants retain the ability to grow on glucose. However, Klpfk2 mutants and the double mutants do not grow on glucose, when respiration is blocked. These data suggest that the pentose phosphate pathway and respiration play a substantial role in glucose utilization by K. lactis. The K. lactis PFK genes can be expressed independently in S. cerevisiae and each of them complements the glucose-negative phenotype of pfk1 pfk2 double deletion mutants in this yeast. Expression of both K. lactis PFK genes simultaneously in S. cerevisiae pfk double deletion mutants complements for PFK activity. However, expression of a combination of PFK genes from K. lactis and S. cerevisiae does not lead to the production of a functional enzyme.

The Primary Structure of Human Liver Type Phosphofructokinase and Its Comparison with Other Types of PFK

The complete mRNA sequence of the human liver-type phosphofructokinase (hPFKL) was determined. The sequence included 55 nucleotides of 5' and 515 of 3' noncoding regions, as well as 2,337 nucleotides encoding the 779 amino acids of the hPFKL. Extensive similarity (approximately 90%) in the coding region was observed between the hPFKL and the mouse PFKL, whereas the degree of similarity between different types of PFK, i.e., hPFKL and human muscle-type PFK (hPFKM), was merely 68%. Nevertheless, striking similarity between these different types of PFK was noticed when the amino acid residues creating the various active sites of the enzyme were compared. Human PFK L- and M-specific probes were constructed and used to quantitate the mRNA levels in fetal and adult brains and fetal liver. It was found that while relative amount of PFKL mRNA in adult brain was one-fourth of that detected in fetal brain the level of PFKM mRNA in adult brain was slightly higher than in fetal tissue, suggesting that PFK expression might be controlled at the transcriptional level.

High-level expression of Bacillus stearothermophilus 6-phosphofructo-l-kinase in Escherichia coli

The 6-phosphofructo-l-kinase (PFK) gene from Bacillus stearothermophilus has been expressed at high levels in Escherichia coli. This expression has been demonstrated by complementation studies, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and PFK assays of cell extracts. A level of B. stearothermophilus PFK expression corresponding to 20% of the total extracted protein was calculated from densitometric scans of an SDS-polyacrylamide gel. The high level of recombinant gene expression will enable this laboratory to determine structure-function relationships in B. stearothermophilus PFK by the method of oligodeoxynucleotide-directed mutagenesis.