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

2021 ◽  
Vol 9 ◽  
Author(s):  
Bin Yang ◽  
Yiwen Sun ◽  
Shouying Fu ◽  
Miaomiao Xia ◽  
Yuan Su ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Carbon Flux ◽  
Parent Strain ◽  
Fold Increase ◽  
Pentose Phosphate ◽  
Growth Defect ◽  
Riboflavin Biosynthesis ◽  
Riboflavin Production ◽  
Single Nucleotide ◽  
Metabolic Phenotypes

Ribulose 5-phosphate (Ru5P) and guanosine 5′-triphosphate (GTP) are two key precursors of riboflavin, whereby Ru5P is also a precursor of GTP. Ribulose 5-phosphate 3-epimerase (Rpe) catalyzes the conversion of ribulose 5-phosphate into xylulose 5-phosphate. Inactivation of Rpe can reduce the consumption of Ru5P, enhancing the carbon flux toward riboflavin biosynthesis. Here we investigated the effect of mutation of rpe and other related genes on riboflavin production, physiological and metabolic phenotypes in Bacillus subtilis LY (BSLY). Introducing single nucleotide deletion (generated BSR) or nonsense mutation (generated BSRN) on the genomic copy of rpe, resulting in more than fivefold increase of riboflavin production over the parental strain. BSR process 62% Rpe activity, while BSRN lost the entire Rpe activity and had a growth defect compared with the parent strain. BSR and BSRN exhibited increases of the inosine and guanine titers, in addition, BSRN exhibited an increase of inosine 5′-monophosphate titer in fermentation. The transcription levels of most oxidative pentose phosphate pathway and purine synthesis genes were unchanged in BSR, except for the levels of zwf and ndk, which were higher than in BSLY. The production of riboflavin was increased to 479.90 ± 33.21 mg/L when ribA was overexpressed in BSR. The overexpression of zwf, gntZ, prs, and purF also enhanced the riboflavin production. 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), showing the potential for further engineering of this strain.

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 CcpN Controls Central Carbon Fluxes in Bacillus subtilis

2008 ◽  
Vol 190 (18) ◽  
pp. 6178-6187 ◽  
Author(s):  
Simon Tännler ◽  
Eliane Fischer ◽  
Dominique Le Coq ◽  
Thierry Doan ◽  
Emmanuel Jamet ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Tricarboxylic Acid Cycle ◽  
Pentose Phosphate ◽  
Growth Defect ◽  
Protein Coding ◽  
Arginine Catabolism ◽  
Futile Cycling ◽  
Mutant Phenotypes ◽  
Tricarboxylic Acid Cycle Intermediates

ABSTRACT The transcriptional regulator CcpN of Bacillus subtilis has been recently characterized as a repressor of two gluconeogenic genes, gapB and pckA, and of a small noncoding regulatory RNA, sr1, involved in arginine catabolism. Deletion of ccpN impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose, gapB and pckA are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of pckA under glycolytic condition causes the growth defect observed in the ccpN mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a ccpN mutant. The high flux through the PP pathway in the ccpN mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of sr1, the third known target of CcpN, plays only a marginal role in ccpN mutant phenotypes.

scholarly journals Metabolic Engineering of Bacillus subtilis for 2.3-BDO production by introducing an exogenous NADPH/NADP+ regeneration system

2019 ◽  
Author(s):  
Huiling Liu ◽  
Shuangying Liu ◽  
Fengyu Xie ◽  
Xian Zhang ◽  
Meijuan Xu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Engineering ◽  
Carbon Flux ◽  
Glucose Dehydrogenase ◽  
Transcriptional Regulator ◽  
Pentose Phosphate ◽  
Regeneration System ◽  
Nadph Regeneration ◽  
By Products ◽  
Regeneration Systems

Abstract Background: Generally, glucose is transformed into pyruvate from glycolysis before the target products acetoin and 2,3-butanediol (2,3-BDO) are formed. Pentose Phosphate Pathway (PPP) is an inefficient synthetic pathway for pyruvate production from glucose in Bacillus subtilis. Previously, it was found that engineered PPP in B. subtilis unbalanced NADH and NADPH regeneration systems and affected acetoin and 2,3 -BDO production.Results: In this study, metabolic engineering strategies were proposed to redistribute carbon flux to 2,3-BDO via reconstructing intracellular cofactors regeneration systems. Firstly, extra copies of glucose dehydrogenase (GDH)and an exogenous NADPH-dependent 2,3-BDO dehydrogenase (TDH) were introduced into the GRAS strain B. subtilis 168 to introduce an exogenous NADPH/NADP + regeneration system and broaden 2,3-BDO production pathway. It was found that overexpressing the NADPH/NADP + regeneration system effectively improved 2,3-BDO production and inhibited NADH-dependent by-products accumulation. Subsequently, the disruption of lactate dehydrogenase (encoded by ldh ) by insertion of the transcriptional regulator ALsR, essential for the expression of alsSD (encoding two key enzymes for the conversion of pyruvate to acetoin) in B. subtilis, resulted in the recombinant strain in which alsSD was overexpressed and the pathway to lactate was blocked simultaneously. On fermentation by the result engineered strain, the highest 2,3-BDO concentration increased by18.43%, while the titers of main byproducts acetoin and lactate decreased by 22.03% and 64%, respectively.Conclusion: In this study, it shows that engineering PPP and reconstructing intracellular cofactors regeneration system could be an alternative strategy in the metabolic engineering of 2,3-BDO production in B. subtilis .

scholarly journals Impact of Activation of the Neotrehalosadiamine/Kanosamine Biosynthetic Pathway on the Metabolism of Bacillus subtilis

2021 ◽  
Vol 203 (9) ◽  
Author(s):  
Natsumi Saito ◽  
Huong Minh Nguyen ◽  
Takashi Inaoka
Keyword(s):  
Bacillus Subtilis ◽  
Energy Metabolism ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Growth Defect ◽  
Metabolome Analysis ◽  
Content Type ◽  
Tca Cycle Intermediates

ABSTRACT The pentose phosphate (PP) pathway is one of the major sources of cellular NADPH. A Bacillus subtilis zwf mutant that lacks glucose-6-phosphate dehydrogenase (the enzyme that catalyzes the first step of the PP pathway) showed inoculum-dose-dependent growth. This growth defect was suppressed by glcP disruption, which causes the upregulation of the autoinducer neotrehalosadiamine (NTD)/kanosamine biosynthetic pathway. A metabolome analysis showed that the stimulation of NTD/kanosamine biosynthesis caused significant accumulation of tricarboxylic acid (TCA) cycle intermediates and NADPH. Because the major malic enzyme YtsJ concomitantly generates NADPH through malate-to-pyruvate conversion, de novo NTD/kanosamine biosynthesis can result in an increase in the intracellular NADPH pool via the accumulation of malate. In fact, a zwf mutant grew in malate-supplemented medium. Artificial induction of glcP in the zwf mutant caused a reduction in the intracellular NADPH pool. Moreover, the correlation between the expression level of the NTD/kanosamine biosynthesis operon ntdABC and the intracellular NADPH pool was confirmed. Our results suggest that NTD/kanosamine has the potential to modulate carbon energy metabolism through an autoinduction mechanism. IMPORTANCE Autoinducers enable bacteria to sense cell density and to coordinate collective behavior. NTD/kanosamine is an autoinducer produced by B. subtilis and several close relatives, although its physiological function remains unknown. The most important finding of this study was the significance of de novo NTD/kanosamine biosynthesis in the modulation of the central carbon metabolism in B. subtilis. We showed that NTD/kanosamine biosynthesis caused an increase in the NADPH pool via the accumulation of TCA cycle intermediates. These results suggest a possible role for NTD/kanosamine in carbon energy metabolism. As Bacillus species are widely used for the industrial production of various useful enzymes and compounds, the NTD/kanosamine biosynthetic pathway might be utilized to control metabolic pathways in these industrial strains.

Improved Production of Two Anti-Candida Lipopeptide Homologues Co- Produced by the Wild-Type Bacillus subtilis RLID 12.1 under Optimized Conditions

2020 ◽  
Vol 21 (5) ◽  
pp. 438-450
Author(s):  
Ramya Ramchandran ◽  
Swetha Ramesh ◽  
Anviksha A ◽  
RamLal Thakur ◽  
Arunaloke Chakrabarti ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Pathogenic Fungi ◽  
Fold Increase ◽  
Production Yield ◽  
Yield Enhancement ◽  
Bioactive Metabolites ◽  
Candida Auris ◽  
Media Formulation ◽  
Static Conditions

Background:: Antifungal cyclic lipopeptides, bioactive metabolites produced by many species of the genus Bacillus, are promising alternatives to synthetic fungicides and antibiotics for the biocontrol of human pathogenic fungi. In a previous study, the co- production of five antifungal lipopeptides homologues (designated as AF1, AF2, AF3, AF4 and AF5) by the producer strain Bacillus subtilis RLID 12.1 using unoptimized medium was reported; though the two homologues AF3 and AF5 differed by 14 Da and in fatty acid chain length were found effective in antifungal action, the production/ yield rate of these two lipopeptides determined by High-Performance Liquid Chromatography was less in the unoptimized media. Methods:: In this study, the production/yield enhancement of the two compounds AF3 and AF5 was specifically targeted. Following the statistical optimization (Plackett-Burman and Box-Behnken designs) of media formulation, temperature and growth conditions, the production of AF3 and AF5 was improved by about 25.8- and 7.4-folds, respectively under static conditions. Results:: To boost the production of these two homologous lipopeptides in the optimized media, heat-inactivated Candida albicans cells were used as a supplement resulting in 34- and 14-fold increase of AF3 and AF5, respectively. Four clinical Candida auris isolates had AF3 and AF5 MICs (100 % inhibition) ranging between 4 and 16 μg/ml indicating the lipopeptide’s clinical potential. To determine the in vitro pharmacodynamic potential of AF3 and AF5, time-kill assays were conducted which showed that AF3 (at 4X and 8X concentrations) at 48h exhibited mean log reductions of 2.31 and 3.14 CFU/ml of C. albicans SC 5314, respectively whereas AF5 at 8X concentration showed a mean log reduction of 2.14 CFU/ml. Conclusion:: With the increasing threat of multidrug-resistant yeasts and fungi, these antifungal lipopeptides produced by optimized method promise to aid in the development of novel antifungal that targets disease-causing fungi with improved efficacy.

scholarly journals TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) Is Upregulated in Lymphocytes Stimulated with Concanavalin A

2021 ◽  
Vol 22 (14) ◽  
pp. 7436
Author(s):  
Helga Simon-Molas ◽  
Xavier Vallvé-Martínez ◽  
Irene Caldera-Quevedo ◽  
Pere Fontova ◽  
Claudia Arnedo-Pac ◽  
...  
Keyword(s):  
Concanavalin A ◽  
Carbon Flux ◽  
Human Lymphocytes ◽  
Tumor Development ◽  
Pentose Phosphate ◽  
Akt Signaling Pathway ◽  
G6pdh Activity ◽  
Physiological Conditions ◽  
Glucose 6 Phosphate Dehydrogenase

The glycolytic modulator TP53-Inducible Glycolysis and Apoptosis Regulator (TIGAR) is overexpressed in several types of cancer and has a role in metabolic rewiring during tumor development. However, little is known about the role of this enzyme in proliferative tissues under physiological conditions. In the current work, we analysed the role of TIGAR in primary human lymphocytes stimulated with the mitotic agent Concanavalin A (ConA). We found that TIGAR expression was induced in stimulated lymphocytes through the PI3K/AKT pathway, since Akti-1/2 and LY294002 inhibitors prevented the upregulation of TIGAR in response to ConA. In addition, suppression of TIGAR expression by siRNA decreased the levels of the proliferative marker PCNA and increased cellular ROS levels. In this model, TIGAR was found to support the activity of glucose 6-phosphate dehydrogenase (G6PDH), the first enzyme of the pentose phosphate pathway (PPP), since the inhibition of TIGAR reduced G6PDH activity and increased autophagy. In conclusion, we demonstrate here that TIGAR is upregulated in stimulated human lymphocytes through the PI3K/AKT signaling pathway, which contributes to the redirection of the carbon flux to the PPP.

scholarly journals Phosphoribosyl pyrophosphate and phosphoribosyl pyrophosphate synthetase in rat mammary gland. Changes in the lactation cycle and effects of diabetes, insulin and phenazine methosulphate

1986 ◽  
Vol 238 (2) ◽  
pp. 553-559 ◽  
Author(s):  
S Kunjara ◽  
M Sochor ◽  
N Salih ◽  
P McLean ◽  
A L Greenbaum
Keyword(s):  
Mammary Gland ◽  
Fold Increase ◽  
Pentose Phosphate ◽  
Diabetic Rat ◽  
Synthetase Activity ◽  
Phosphoribosyl Pyrophosphate ◽  
Rat Mammary Gland ◽  
Lactation Cycle ◽  
Phenazine Methosulphate

Changes in the tissue content of phosphoribosyl pyrophosphate (PPRibP), glucose 6-phosphate, ribose 5-phosphate (Rib5P), RNA and DNA, of the activity of PPRibP synthetase (EC 2.7.6.1) and the conversion of [1-14C]- and [6-14C]-glucose into 14CO2 were measured at mid-lactation in the normal and diabetic rat and in pregnancy, lactation and mammary involution in the normal rat. The PPRibP, glucose 6-phosphate and Rib5P contents increase during pregnancy and early lactation to reach a plateau value at mid-lactation, before falling sharply during weaning. The PPRibP content, PPRibP synthetase activity and flux of glucose through the oxidative pentose phosphate pathway (PPP) all change in parallel during the lactation cycle. Similarly, after 3 and 5 days duration of streptozotocin-induced diabetes, ending on day 10 of lactation, there were parallel declines in PPRibP content, PPRibP synthetase and PPP activity. The effect of streptozotocin was prevented by pretreatment with nicotinamide and partially reversed by insulin administration. Addition of insulin to lactating rat mammary-gland slices incubated in vitro significantly raised the PPRibP content (+47%) and the activity of the PPP (+40%); phenazine methosulphate, which gives a 2-fold increase in PPP activity, raised the PPRibP content of lactating mammary gland slices by approx. 3-fold. It is concluded that Rib5P, generated in the oxidative segment of the PPP, is an important determinant of PPRibP synthesis in the lactating rat mammary gland and that insulin plays a central role in the regulation of the bioavailability of this precursor of nucleotide and nucleic acid synthesis.

scholarly journals The Bacillus subtilis yqjI Gene Encodes the NADP+-Dependent 6-P-Gluconate Dehydrogenase in the Pentose Phosphate Pathway

2004 ◽  
Vol 186 (14) ◽  
pp. 4528-4534 ◽  
Author(s):  
Nicola Zamboni ◽  
Eliane Fischer ◽  
Dietmar Laudert ◽  
Stéphane Aymerich ◽  
Hans-Peter Hohmann ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Glucose Dehydrogenase ◽  
Reducing Power ◽  
Pentose Phosphate ◽  
Gene Encoding ◽  
Metabolic Intermediates ◽  
The Third ◽  
Key Enzyme ◽  
Sequence Similarities

ABSTRACT Despite the importance of the oxidative pentose phosphate (PP) pathway as a major source of reducing power and metabolic intermediates for biosynthetic processes, almost no direct genetic or biochemical evidence is available for Bacillus subtilis. Using a combination of knockout mutations in known and putative genes of the oxidative PP pathway and 13C-labeling experiments, we demonstrated that yqjI encodes the NADP+-dependent 6-P-gluconate dehydrogenase, as was hypothesized previously from sequence similarities. Moreover, YqjI was the predominant isoenzyme during glucose and gluconate catabolism, and its role in the oxidative PP pathway could not be played by either of two homologues, GntZ and YqeC. This conclusion is in contrast to the generally held view that GntZ is the relevant isoform; hence, we propose a new designation for yqjI, gndA, the monocistronic gene encoding the principal 6-P-gluconate dehydrogenase. Although we demonstrated the NAD+-dependent 6-P-gluconate dehydrogenase activity of GntZ, gntZ mutants exhibited no detectable phenotype on glucose, and GntZ did not contribute to PP pathway fluxes during growth on glucose. Since gntZ mutants grew normally on gluconate, the functional role of GntZ remains obscure, as does the role of the third homologue, YqeC. Knockout of the glucose-6-P dehydrogenase-encoding zwf gene was primarily compensated for by increased glycolytic fluxes, but about 5% of the catabolic flux was rerouted through the gluconate bypass with glucose dehydrogenase as the key enzyme.

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