Enhancement of riboflavin production in Bacillus subtilis via in vitro and in vivo metabolic engineering of pentose phosphate pathway

2021 ◽  
Author(s):  
Mengxue Zhang ◽  
Xingcong Zhao ◽  
Xi Chen ◽  
Mingyue Li ◽  
Xuedong Wang
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Engineering ◽  
Pentose Phosphate Pathway ◽  
Pentose Phosphate ◽  
Riboflavin Production

scholarly journals Beneficial effects of acute inhibition of the oxidative pentose phosphate pathway in the failing heart

2014 ◽  
Vol 306 (5) ◽  
pp. H709-H717 ◽  
Author(s):  
Claudio Vimercati ◽  
Khaled Qanud ◽  
Gianfranco Mitacchione ◽  
Danuta Sosnowska ◽  
Zoltan Ungvari ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pentose Phosphate Pathway ◽  
Tissue Concentration ◽  
Glucose Consumption ◽  
Pentose Phosphate ◽  
Oxidative Pentose Phosphate Pathway ◽  
Stroke Work ◽  
Failing Heart

In vitro studies suggested that glucose metabolism through the oxidative pentose phosphate pathway (oxPPP) can paradoxically feed superoxide-generating enzymes in failing hearts. We therefore tested the hypothesis that acute inhibition of the oxPPP reduces oxidative stress and enhances function and metabolism of the failing heart, in vivo. In 10 chronically instrumented dogs, congestive heart failure (HF) was induced by high-frequency cardiac pacing. Myocardial glucose consumption was enhanced by raising arterial glycemia to levels mimicking postprandial peaks, before and after intravenous administration of the oxPPP inhibitor 6-aminonicotinamide (80 mg/kg). Myocardial energy substrate metabolism was measured with radiolabeled glucose and oleic acid, and cardiac 8-isoprostane output was used as an index of oxidative stress. A group of five chronically instrumented, normal dogs served as control. In HF, raising glycemic levels from ∼80 to ∼170 mg/dL increased cardiac isoprostane output by approximately twofold, whereas oxPPP inhibition normalized oxidative stress and enhanced cardiac oxygen consumption, glucose oxidation, and stroke work. In normal hearts glucose infusion did not induce significant changes in cardiac oxidative stress. Myocardial tissue concentration of 6P-gluconate, an intermediate metabolite of the oxPPP, was significantly reduced by ∼50% in treated versus nontreated failing hearts, supporting the inhibitory effect of 6-aminonicotinamide. Our study indicates an important contribution of the oxPPP activity to cardiac oxidative stress in HF, which is particularly pronounced during common physiological changes such as postprandial glycemic peaks.

scholarly journals The pentose phosphate pathway regulates chronic neuroinflammation and dopaminergic neurodegeneration

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Dezhen Tu ◽  
Yun Gao ◽  
Ru Yang ◽  
Tian Guan ◽  
Jau-Shyong Hong ◽  
...  
Keyword(s):  
Substantia Nigra ◽  
Pentose Phosphate Pathway ◽  
Microglial Activation ◽  
Redox Homeostasis ◽  
Pentose Phosphate ◽  
Transgenic Expression ◽  
Dopaminergic Neurodegeneration ◽  
Locomotor Impairment

Abstract Background Metabolic dysfunction and neuroinflammation are increasingly implicated in Parkinson’s disease (PD). The pentose phosphate pathway (PPP, a metabolic pathway parallel to glycolysis) converts glucose-6-phosphate into pentoses and generates ribose-5-phosphate and NADPH thereby governing anabolic biosynthesis and redox homeostasis. Brains and immune cells display high activity of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP. A postmortem study reveals dysregulation of G6PD enzyme in brains of PD patients. However, spatial and temporal changes in activity/expression of G6PD in PD remain undetermined. More importantly, it is unclear how dysfunction of G6PD and the PPP affects neuroinflammation and neurodegeneration in PD. Methods We examined expression/activity of G6PD and its association with microglial activation and dopaminergic neurodegeneration in multiple chronic PD models generated by an intranigral/intraperitoneal injection of LPS, daily subcutaneous injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 6 days, or transgenic expression of A53T α-synuclein. Primary microglia were transfected with G6PD siRNAs and treated with lipopolysaccharide (LPS) to examine effects of G6PD knockdown on microglial activation and death of co-cultured neurons. LPS alone or with G6PD inhibitor(s) was administrated to mouse substantia nigra or midbrain neuron-glia cultures. While histological and biochemical analyses were conducted to examine microglial activation and dopaminergic neurodegeneration in vitro and in vivo, rotarod behavior test was performed to evaluate locomotor impairment in mice. Results Expression and activity of G6PD were elevated in LPS-treated midbrain neuron-glia cultures (an in vitro PD model) and the substantia nigra of four in vivo PD models. Such elevation was positively associated with microglial activation and dopaminergic neurodegeneration. Furthermore, inhibition of G6PD by 6-aminonicotinamide and dehydroepiandrosterone and knockdown of microglial G6PD attenuated LPS-elicited chronic dopaminergic neurodegeneration. Mechanistically, microglia with elevated G6PD activity/expression produced excessive NADPH and provided abundant substrate to over-activated NADPH oxidase (NOX2) leading to production of excessive reactive oxygen species (ROS). Knockdown and inhibition of G6PD ameliorated LPS-triggered production of ROS and activation of NF-кB thereby dampening microglial activation. Conclusions Our findings indicated that G6PD-mediated PPP dysfunction and neuroinflammation exacerbated each other mediating chronic dopaminergic neurodegeneration and locomotor impairment. Insight into metabolic-inflammatory interface suggests that G6PD and NOX2 are potential therapeutic targets for PD.

scholarly journals Improving the Production of Riboflavin by Introducing a Mutant Ribulose 5-Phosphate 3-Epimerase Gene in Bacillus Subtilis

2021 ◽  
Author(s):  
Bin Yang ◽  
Yiwen Sun ◽  
Shouying Fu ◽  
Miaomiao Xia ◽  
Chuan Liu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Pentose Phosphate Pathway ◽  
De Novo ◽  
Fold Increase ◽  
Fermentation Medium ◽  
Pentose Phosphate ◽  
Oxidative Pentose Phosphate Pathway ◽  
Growth Defect ◽  
Riboflavin Production ◽  
Expression Of Genes

Abstract Background: Ribulose 5-phosphate (Ru5P) and guanosine 5'-triphosphate (GTP) are two key precursors of riboflavin, whereby Ru5P is also a precursor of GTP (purine de novo synthesis pathway). As a product of the oxidative pentose phosphate pathway (OPPP), Ru5P can flow back into the Embden-Meyerhof-Parnas pathway (EMP) through the non-oxidative pentose phosphate pathway (NOPPP). Major enzymes of the NOPPP include transaldolase (Tal), transketolase (Tkt), and ribulose 5-phosphate 3-epimerase (Rpe), which catalyze the conversion of Ru5P into xylulose 5-phosphate (X5P). Thus, inactivation of Rpe can reduce the consumption of Ru5P, enhancing the carbon flux toward riboflavin biosynthesis. However, there are no studies on the mutation or deletion of Rpe for improving riboflavin production. Results: We mutated the genomic copy of rpe, in the riboflavin-producing Bacillus subtilis BSLY. The resulting strain BSR produced 320.50±31.28 mg/L of riboflavin in a fermentation medium containing 40 g/L sucrose as the carbon source, representing a 5-fold increase over the parental strain. We also constructed an rpe nonsense mutation strain BSRN, which produced 365.69±27.53 mg/L riboflavin in fermentation medium. However, BSRN had a growth defect compared with BSR and BSLY, which was also present in media containing glucose, maltose, and fructose. The growth of BSRN was restored when mutant or wild-type rpe was overexpressed. Notably, BSR exhibited respective increases of the inosine and guanine titers by 163% and 40%. Furthermore, a 35.96-fold increase of inosine 5’-monophosphate (IMP) titer was detected in BSRN. The transcription levels of most OPPP, purine, and GTP synthesis genes were unchanged in BSR, except for the levels of zwf and ndk, which were respectively 49% lower and 310% higher than in BSLY. The production of riboflavin was increased to 479.90±33.21 mg/L when ribA was expressed in BSR. The expression of zwf, gntZ, prs, and purF, respectively enhanced the riboflavin production by 48%, 31%, 26%, and 35%. Finally, overexpression of the rib operon by the pMX45 plasmid and mutant gnd by pHP03 plasmid in BSR led to a 3.05-fold increase of the riboflavin production (977.29±63.44 mg/L).Conclusions: This paper describes a mutation of the ribulose 5-phosphate 3-epimerase and its influence on riboflavin production. The results of qRT-PCR and HPLC analyses indicated that the rpe mutant showed a different pattern of purine metabolism, while the cells maintained generally normal levels of the transcription of genes related to PPP and purine de novel synthesis pathways. With the increased expression of genes in the OPPP, purine, and riboflavin synthesis pathways, the production of riboflavin was effectively enhanced, showing the potential for further engineering of this strain.

scholarly journals Induction of a Futile Embden-Meyerhof-Parnas Pathway in Deinococcus radiodurans by Mn: Possible Role of the Pentose Phosphate Pathway in Cell Survival

2000 ◽  
Vol 66 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Y.-M. Zhang ◽  
T.-Y. Wong ◽  
L.-Y. Chen ◽  
C.-S. Lin ◽  
J.-K. Liu
Keyword(s):  
Pentose Phosphate Pathway ◽  
Statistical Models ◽  
Deinococcus Radiodurans ◽  
Uv Light ◽  
Pentose Phosphate ◽  
Marginal Effect ◽  
Biomass Formation

ABSTRACT Statistical models were used to predict the effects of tryptone, glucose, yeast extract (TGY) and Mn on biomass formation of the highly radioresistant bacterium Deinococcus radiodurans. Results suggested that glucose had marginal effect on biomass buildup, but Mn was a significant factor for biomass formation. Mn also facilitated glucose interactions with other nutrient components. These predictions were verified by in vivo and in vitro experiments. TGY-grown cells metabolized glucose solely by the pentose phosphate pathway (PPP). Although only a fraction of glucose from the medium was transported into the cells, glucose was incorporated into the DNA efficiently after cells were exposed to UV light. The presence of glucose also enhanced the radioresistance of the culture. Mn could induce an Embden-Meyerhof-Parnas (EMP) pathway in D. radiodurans. The EMP pathway and the PPP of the Mn-treated cells oxidized glucose simultaneously at a 6:1 ratio. Although glucose was hydrolyzed rapidly by the Mn-treated cells, most glucose was released as CO2. Mn-treated cultures retained less glucose per cell than cells grown without Mn, and still less glucose was incorporated into the DNA after cells were exposed to UV light. Mn-treated cells were also more sensitive to UV light. Results suggested that metabolites of glucose generated from the PPP enhanced the survival of D. radiodurans. Induction of the EMP pathway by Mn may deplete metabolites for DNA repair and may induce oxidative stress for the cell, leading to reduction of radioresistance.

scholarly journals PFKFB4 is overexpressed in clear-cell renal cell carcinoma promoting pentose phosphate pathway that mediates Sunitinib resistance

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Chenchen Feng ◽  
Yuqing Li ◽  
Kunping Li ◽  
Yinfeng Lyu ◽  
Wenhui Zhu ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Pentose Phosphate Pathway ◽  
Renal Cell ◽  
Clear Cell ◽  
Pentose Phosphate ◽  
Fine Tuning ◽  
Cell Renal Cell Carcinoma

Abstract Background Kinases play critical role in clear-cell renal cell carcinoma (ccRCC). We aim to exploit novel kinase that is both protumorigenic and drugable in ccRCC. Methods Reproduction of public datasets with validation using microarray was performed to identify candidate gene. Functionality was studied using multi-omics with validation in vitro and in vivo. Results 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 (PFKFB4) was differentially expressed showing significantly higher expression in tumor than in normal kidney. PFKFB4 overexpression was associated with advanced tumor grade, stage and worsened prognosis. PFKFB4-knockdown significantly impaired fitness in cell proliferation, migration and wound healing. Despite being recurrently deleted on 3p, PFKFN4 mRNA remained actively transcribed by HIF1α. Metabolomics showed overexpressed PFKFB4 showed enriched metabolites in pentose phosphate pathway (PPP). Phosphoproteomics and immunoprecipitation showed PFKFB4 also phosphorylated NCOA3 which interacted with FBP1 to counteract overactive PPP flux, forming a regulatory loop. PFKFB4-knockdown overcame resistance to Sunitinib in vitro and in vivo both in xenograft and tail-vein injection murine models. Conclusion We concluded PFKFB4 was associated with PPP activity and the fine-tuning of which was mediated by its phosphorylation of NCOA3. Targeting PFKFB4 held promise to combat resistance to Sunitinib.

scholarly journals In Bacillus subtilis, the Sirtuin Protein Deacetylase, Encoded by the srtN Gene (Formerly yhdZ), and Functions Encoded by the acuABC Genes Control the Activity of Acetyl Coenzyme A Synthetase

2009 ◽  
Vol 191 (6) ◽  
pp. 1749-1755 ◽  
Author(s):  
Jeffrey G. Gardner ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Bacillus Subtilis ◽  
Coenzyme A ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
New Information ◽  
Low Concentrations ◽  
Dependent Protein ◽  
Protein Deacetylase

ABSTRACT This report provides in vivo evidence for the posttranslational control of the acetyl coenzyme A (Ac-CoA) synthetase (AcsA) enzyme of Bacillus subtilis by the acuA and acuC gene products. In addition, both in vivo and in vitro data presented support the conclusion that the yhdZ gene of B. subtilis encodes a NAD+-dependent protein deacetylase homologous to the yeast Sir2 protein (also known as sirtuin). On the basis of this new information, a change in gene nomenclature, from yhdZ to srtN (for sirtuin), is proposed to reflect the activity associated with the YdhZ protein. In vivo control of B. subtilis AcsA function required the combined activities of AcuC and SrtN. Inactivation of acuC or srtN resulted in slower growth and cell yield under low-acetate conditions than those of the wild-type strain, and the acuC srtN strain grew under low-acetate conditions as poorly as the acsA strain. Our interpretation of the latter result was that both deacetylases (AcuC and SrtN) are needed to maintain AcsA as active (i.e., deacetylated) so the cell can grow with low concentrations of acetate. Growth of an acuA acuC srtN strain on acetate was improved over that of the acuA + acuC srtN strain, indicating that the AcuA acetyltransferase enzyme modifies (i.e., inactivates) AcsA in vivo, a result consistent with previously reported in vitro evidence that AcsA is a substrate of AcuA.

scholarly journals ROS/PI3K/Akt and Wnt/β-catenin signalings activate HIF-1α-induced metabolic reprogramming to impart 5-fluorouracil resistance in colorectal cancer

2022 ◽  
Vol 41 (1) ◽  
Author(s):  
Shuohui Dong ◽  
Shuo Liang ◽  
Zhiqiang Cheng ◽  
Xiang Zhang ◽  
Li Luo ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Glucose Metabolism ◽  
Pentose Phosphate ◽  
Metabolic Reprogramming ◽  
Cell Models ◽  
Primary Tumors ◽  
Pharmacological Inhibition ◽  
Potential Biomarker

Abstract Background Acquired resistance of 5-fluorouracil (5-FU) remains a clinical challenge in colorectal cancer (CRC), and efforts to develop targeted agents to reduce resistance have not yielded success. Metabolic reprogramming is a key cancer hallmark and confers several tumor phenotypes including chemoresistance. Glucose metabolic reprogramming events of 5-FU resistance in CRC has not been evaluated, and whether abnormal glucose metabolism could impart 5-FU resistance in CRC is also poorly defined. Methods Three separate acquired 5-FU resistance CRC cell line models were generated, and glucose metabolism was assessed by measuring glucose and lactate utilization, RNA and protein expressions of glucose metabolism-related enzymes and changes of intermediate metabolites of glucose metabolite pool. The protein levels of hypoxia inducible factor 1α (HIF-1α) in primary tumors and circulating tumor cells of CRC patients were detected by immunohistochemistry and immunofluorescence. Stable HIF1A knockdown in cell models was established with a lentiviral system. The influence of both HIF1A gene knockdown and pharmacological inhibition on 5-FU resistance in CRC was evaluated in cell models in vivo and in vitro. Results The abnormality of glucose metabolism in 5-FU-resistant CRC were described in detail. The enhanced glycolysis and pentose phosphate pathway in CRC were associated with increased HIF-1α expression. HIF-1α-induced glucose metabolic reprogramming imparted 5-FU resistance in CRC. HIF-1α showed enhanced expression in 5-FU-resistant CRC cell lines and clinical specimens, and increased HIF-1α levels were associated with failure of fluorouracil analog-based chemotherapy in CRC patients and poor survival. Upregulation of HIF-1α in 5-FU-resistant CRC occurred through non-oxygen-dependent mechanisms of reactive oxygen species-mediated activation of PI3K/Akt signaling and aberrant activation of β-catenin in the nucleus. Both HIF-1α gene knock-down and pharmacological inhibition restored the sensitivity of CRC to 5-FU. Conclusions HIF-1α is a potential biomarker for 5-FU-resistant CRC, and targeting HIF-1a in combination with 5-FU may represent an effective therapeutic strategy in 5-FU-resistant CRC.

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