Coexistence of the Entner–Doudoroff and Embden–Meyerhof–Parnas pathways enhances glucose consumption of ethanol-producing Corynebacterium glutamicum

Background It is interesting to modify sugar metabolic pathways to improve the productivity of biocatalysts that convert sugars to value-added products. However, this attempt often fails due to the tight control of the sugar metabolic pathways. Recently, activation of the Entner–Doudoroff (ED) pathway in Escherichia coli has been shown to enhance glucose consumption, though the mechanism underlying this phenomenon is poorly understood. In the present study, we investigated the effect of a functional ED pathway in metabolically engineered Corynebacterium glutamicum that metabolizes glucose via the Embden–Meyerhof–Parnas (EMP) pathway to produce ethanol under oxygen deprivation. This study aims to provide further information on metabolic engineering strategies that allow the Entner–Doudoroff and Embden–Meyerhof–Parnas pathways to coexist. Results Three genes (zwf, edd, and eda) encoding glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase from Zymomonas mobilis were expressed in a genetically modified strain, C. glutamicum CRZ2e, which produces pyruvate decarboxylase and alcohol dehydrogenase from Z. mobilis. A 13C-labeling experiment using [1-13C] glucose indicated a distinctive 13C distribution of ethanol between the parental and the ED-introduced strains, which suggested an alteration of carbon flux as a consequence of ED pathway introduction. The ED-introduced strain, CRZ2e-ED, consumed glucose 1.5-fold faster than the parental strain. A pfkA deletion mutant of CRZ2e-ED (CRZ2e-EDΔpfkA) was also constructed to evaluate the effects of EMP pathway inactivation, which showed an almost identical rate of glucose consumption compared to that of the parental CRZ2e strain. The introduction of the ED pathway did not alter the intracellular NADH/NAD+ ratio, whereas it resulted in a slight increase in the ATP/ADP ratio. The recombinant strains with simultaneous overexpression of the genes for the EMP and ED pathways exhibited the highest ethanol productivity among all C. glutamicum strains ever constructed. Conclusions The increased sugar consumption observed in ED-introduced strains was not a consequence of cofactor balance alterations, but rather the crucial coexistence of two active glycolytic pathways for enhanced glucose consumption. Coexistence of the ED and EMP pathways is a good strategy for improving biocatalyst productivity even when NADPH supply is not a limiting factor for fermentation.

CFRP Origami Metamaterial with Tunable Buckling Loads: A Numerical Study

Origami has played an increasingly central role in designing a broad range of novel structures due to its simple concept and its lightweight and extraordinary mechanical properties. Nonetheless, most of the research focuses on mechanical responses by using homogeneous materials and limited studies involving buckling loads. In this study, we have designed a carbon fiber reinforced plastic (CFRP) origami metamaterial based on the classical Miura sheet and composite material. The finite element (FE) modelling process’s accuracy is first proved by utilizing a CFRP plate that has an analytical solution of the buckling load. Based on the validated FE modelling process, we then thoroughly study the buckling resistance ability of the proposed CFRP origami metamaterial numerically by varying the folding angle, layer order, and material properties, finding that the buckling loads can be tuned to as large as approximately 2.5 times for mode 5 by altering the folding angle from 10° to 130°. With the identical rate of increase, the shear modulus has a more significant influence on the buckling load than Young’s modulus. Outcomes reported reveal that tunable buckling loads can be achieved in two ways, i.e., origami technique and the CFRP material with fruitful design freedoms. This study provides an easy way of merely adjusting and controlling the buckling load of lightweight structures for practical engineering.

Stimulation of splanchnic glucose production during exercise in humans contains a glucagon-independent component

To determine the importance of basal glucagon to the stimulation of net splanchnic glucose output (NSGO) during exercise, seven healthy males performed cycle exercise during a pancreatic islet cell clamp. In one group (BG), glucagon was replaced at basal levels and insulin was adjusted to achieve euglycemia. In another group (GD), only insulin was replaced at the identical rate used in BG, and basal glucagon was not replaced. Exogenous glucose infusion was necessary to maintain euglycemia during exercise in BG and during rest and exercise in GD. Arterial glucagon was at least twofold greater in BG than in GD throughout the pancreatic islet cell clamp. Although basal NSGO remained stable in BG (2.5 ± 0.5 mg · kg−1 · min−1), basal NSGO dropped by 70% in GD (0.7 ± 0.3 mg · kg−1 · min−1). NSGO was also greater in BG than in GD at 10 min of moderate exercise, most likely due to the residual effect of basal glucagon replacement. However, NSGO increased slightly and remained similar throughout the remainder of moderate and heavy exercise in BG and GD. Therefore, a mechanism independent of changes in pancreatic hormones and/or the level of glycemia contributes toward modest stimulation of NSGO during moderate and heavy exercise.

Investigation of the slow inhibition of almond β-glucosidase and yeast isomaltase by 1-azasugar inhibitors: evidence for the ‘direct binding’ model

(-)-1-Azafagomine [(3R,4R,5R)-4,5-dihydroxy-3-hydroxymethylhexahydropyridazine; inhibitor 1] is a potent glycosidase inhibitor designed to mimic the transition state of a substrate undergoing glycoside cleavage. The inhibition of glycosidases by inhbitor 1 and analogues has been found to be a relatively slow process. This ‘slow inhibition’ process was investigated in the inhibition of almond β-glucosidase and yeast isomaltase by inhibitor 1 and analogues. Progress-curve experiments established that the time-dependent inhibition of both enzymes by inhibitor 1 was a consequence of relatively slow dissociation and association of the inhibitor from and to the enzyme, and not a result of slow interchanges between protein conformations. A number of hydrazine-containing analogues of inhibitor 1 also inhibited β-glucosidase and isomaltase slowly, while the amine isofagomine [(3R,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine; inhibitor 5] only inhibited β-glucosidase slowly. Inhibitor 1 and related inhibitors were found to leave almond β-glucosidase with almost identical rate constants, so that the difference in Ki values depended almost entirely on changes in the binding rate constant, kon. The same trend was observed for the inhibition of yeast isomaltase by inhibitor 1 and a related inhibitor. The values of the rate constants were obtained at 25 °C and at pH 6.8.

Minimizing expected makespans on uniform processor systems

We study the problem of scheduling n given jobs on m uniform processors to minimize expected makespan (maximum finishing time). Job execution times are not known in advance, but are known to be exponentially distributed, with identical rate parameters depending solely on the executing processor. For m = 2 and 3, we show that there exist optimal scheduling rules of a certain threshold type, and we show how the required thresholds can be easily determined. We conjecture that similar threshold rules suffice for m > 3 but are unable to prove this. However, for m > 3 we do obtain a general bound on problem size that permits Bellman equations to be used to construct an optimal scheduling rule for any given set of m rate parameters, with the memory required to represent that scheduling rule being independent of the number of remaining jobs.

Minimizing expected makespans on uniform processor systems

We study the problem of scheduling n given jobs on m uniform processors to minimize expected makespan (maximum finishing time). Job execution times are not known in advance, but are known to be exponentially distributed, with identical rate parameters depending solely on the executing processor. For m = 2 and 3, we show that there exist optimal scheduling rules of a certain threshold type, and we show how the required thresholds can be easily determined. We conjecture that similar threshold rules suffice for m > 3 but are unable to prove this. However, for m > 3 we do obtain a general bound on problem size that permits Bellman equations to be used to construct an optimal scheduling rule for any given set of m rate parameters, with the memory required to represent that scheduling rule being independent of the number of remaining jobs.

The Role of an Anaerobic Stage on Biological Phosphorus Removal

A detailed study was conducted on biological removal of phosphorus by an activated sludge process composed of an anaerobic stage followed by an aerobic stage in order to clarify the role of the anaerobic stage of the process. Two distinctive sludges, one contained approximately 10 percent phosphorus, most of which existed in the form of polyphosphates, and the other contained 1.9 percent phosphorus, were obtained by changing BOD concentration of influent and hydraulic detention time of the process. Although the polyphosphate-containing sludge released phosphorus in proportion to the absorbed BOD at anaerobic conditions, the sludge which did not contain polyphosphates also absorbed an equal amount of BOD at the identical rate without any external sources of oxygen. Both sludges accumulated identical levels of poly-β-hydroxybutyrate within the sludges as the BOD sources were absorbed. These results suggest that the polyphosphate-containing sludges have no advantage over the sludges with no polyphosphates when they uptake BOD in the anaerobic stage.

A study of the properties of two porphyringlobin species formed in the reaction of protoporphyrin IX with human globin

Globin was prepared from the main (A0) component of human haemoglobin and reacted with protoporphyrin IX; the product, when subjected to chromatography on CM-Sephadex, separated into fast- and slow-moving species. These were isolated for examination. The dissociation constant for the tetramer–dimer equilibrium of fast-moving porphyringlobin was determined at 2.8X10−6m; this is to be compared with values of 2.2X10−6m and 8X10−8m determined for oxyhaemoglobin and the slow-moving porphyringlobin respectively. It was also shown that the thiol groups of fast-moving porphyringlobin react with 4,4′dithiodipyridine at an identical rate with those of oxyhaemoglobin; in comparison, the rates of reaction of deoxyhaemoglobin and porphyringlobin are much slower but are again identical with one another. The quenching of porphyringlobin fluorescence by I− ions was also studied. The quenching could not be represented by a simple Stern–Volmer relationship (whereas that of porphyrin–apomyoglobin is), but was represented by a model in which the fluorescence of fast-moving porphyringlobin was more accessible to the quencher than that of the slow-moving component. Similarly, fast-moving porphyringlobin was photodecomposed more rapidly by oxygen than the slow-moving species.