Metabolic regulation inferred from Jacobian and Hessian matrices of metabolic functions

Quantitative analysis of experimental metabolic data is frequently challenged by non-intuitive, complex patterns which emerge from regulatory networks. Quantitative output of metabolic regulation can be summarised by metabolic functions which comprise information about dynamics of metabolite concentrations. They reflect the sum of biochemical reactions which affect a metabolite concentration. Derivatives of metabolic functions provide essential information about system dynamics. The Jacobian matrix of a reaction network summarises first-order partial derivatives of metabolic functions with respect to metabolite concentrations while Hessian matrices summarise second-order partial derivatives. Here, a simple model of invertase-driven sucrose hydrolysis is simulated and both Jacobian and Hessian matrices of metabolic functions are derived for quantitative analysis of kinetic regulation of sucrose metabolism. Based on previous experimental observations, metabolite dynamics are quantitatively explained in context of underlying metabolic functions. Their potential regulatory role during plant cold acclimation is derived from Jacobian and Hessian matrices.

Comparison of Spathaspora passalidarum and recombinant Saccharomyces cerevisiae for integration of first- and second-generation ethanol production

ABSTRACT First-generation ethanol (E1G) is based on the fermentation of sugars released from saccharine or starch sources, while second-generation ethanol (E2G) is focused on the fermentation of sugars released from lignocellulosic feedstocks. During the fractionation process to release sugars from hemicelluloses (mainly xylose), some inhibitor compounds are released hindering fermentation. Thus, the biggest challenge of using hemicellulosic hydrolysate is selecting strains and processes able to efficiently ferment xylose and tolerate inhibitors. With the aim of diluting inhibitors, sugarcane molasses (80% of sucrose content) can be mixed to hemicellulosic hydrolysate in an integrated E1G–E2G process. Cofermentations of xylose and sucrose were evaluated for the native xylose consumer Spathaspora passalidarum and a recombinant Saccharomyces cerevisiae strain. The industrial S. cerevisiae strain CAT-1 was modified to overexpress the XYL1, XYL2 and XKS1 genes and a mutant ([4–59Δ]HXT1) version of the low-affinity HXT1 permease, generating strain MP-C5H1. Although S. passalidarum showed better results for xylose fermentation, this yeast showed intracellular sucrose hydrolysis and low sucrose consumption in microaerobic conditions. Recombinant S. cerevisiae showed the best performance for cofermentation, and a batch strategy at high cell density in bioreactor achieved unprecedented results of ethanol yield, titer and volumetric productivity in E1G–E2G production process.

Sucrose Hydrolysis in a Continuous Packed-Bed Reactor with Auto-Immobilised Aspergillus Niger Biocatalyst Obtained by Solid-State Fermentation

Invertase from Aspergillus niger C28B25 was produced by solid-state fermentation (SSF), and fermented solids were used directly as a biocatalyst for batch and continuous hydrolysis of sucrose in a packed-bed reactor under different operational conditions with varied temperature, sucrose concentration and feed flow rate. The SSF allowed obtaining a biocatalyst with an invertase activity of 82.2 U/g db. The biocatalyst maintained its activity in the range of 40 to 70ºC for at least 70 h of continuous operation. The highest hydrolysis rate (12.3 mg/U h) was obtained at 70ºC with 2M sucrose under batch conditions. Continuous hydrolysis in 20-mL and 200-mL bioreactors at 60ºC led to sucrose hydrolysis above 60% (8.5 residence times) and above 55% (4.5 residence times), respectively. The auto-immobilised biocatalyst produced by SSF without recovery, purification and immobilisation stages offers an economical alternative for the development of accessible biocatalysts that can be applied in batch or continuous sucrose hydrolysis processes. This study shows the potential of biocatalyst production by SSF for other enzymatic systems.

An Optical Chiral Sensor Based on Weak Measurement for the Real-Time Monitoring of Sucrose Hydrolysis

A chiral sensor with optical rotation detection based on weak measurement for the kinetic study of sucrose hydrolysis is presented. Based on the polarization modulation to the pre-selection state, the optical rotation of chiral sample was accurately determined through the central wavelength shift of the output spectrum. With this approach, the concentration response curves of sucrose and its hydrolysis products, i.e., fructose and glucose, were experimentally obtained for the hydrolysis analysis. By collecting the output spectrum with a frequency of 100 Hz and fitting the central wavelength shift synchronously during the measurement, the sucrose hydrolysis process was monitored in real time. Different hydrolysis conditions with varied concentration of invertase enzyme and citrate were implemented in this work. As a consequence, the real-time hydrolysis curves of the hydrolysis process with distinct velocities was achieved and analyzed. Such a kinetic monitoring about sucrose hydrolysis with optical rotation detection technology played a critical role in the researches involving sucrose, and also revealed the great potential of weak measurement in intersections, such as food safety inspection and chemical analysis.

Optimization of β-Fructofuranosidase Production from Agrowaste by Aspergillus carbonarius and Its Application in the Production of Inverted Sugar

Research background. Microbial β-fructofuranosidases are widely employed in food industry to produce inverted sugar or fructooligosaccharides. In this study, a newly isolated Aspergillus carbonarius PC-4 strain was used to optimize the β-fructofuranosidase production under a cost-effective process and the sucrose hydrolysis was evaluated to produce inverted sugars. Experimental approach. Optimization of nutritional components of culture medium was carried out using Simplex Lattice mixture design for 72 h and 120 h at 28 ˚C. One-Factor-at-a-time methodology was used to optimize the physicochemical parameters. Crude enzyme was used for sucrose hydrolysis at different concentrations. Results and conclusions. The optimized condition of enzyme production was pineapple crown waste (1.3 %, m/V) and yeast extract (0.3 %, m/V) for 72 h (9.4 U/mL), obtaining R² 91.85 %, R² adjusted 85.06 %, highest F value (13.52) and low p-value (0.003). One-factor-at-a-time used for optimizing the physicochemical conditions showed optimum temperature (20 ˚C), pH (5.5), agitation (180 rpm) and time-course (72 h) with an increase of 3.0-folds for enzyme production. The invertase-induced sucrose hydrolysis showed the maximum yield (3,451.7 µmols of reducing sugars) using 10 % of initial sucrose concentration. Higher sucrose concentrations caused inhibition of invertase activity, possibly due to saturation of substrate or formation of sucrose aggregates making it difficult for the enzyme to access sucrose molecules within the created clusters. So, a cost-effective method was developed for the invertase production using agroindustrial waste and the enzyme produced can be used efficiently for inverted sugar production at high sucrose concentration. Novelty and scientific contribution. This study presents an efficient utilization of pineapple crown wastes to produce invertase by a newly isolated Aspergillus carbonarius PC-4 strain. This enzyme exhibited a good potential for invert sugar production at high initial sucrose concentration, which is interesting for industrial applications.

Restriction of cytosolic sucrose hydrolysis profoundly alters development, metabolism and gene expression in Arabidopsis roots

Plant roots depend on sucrose imported from leaves as the substrate for metabolism and growth. Sucrose and hexoses derived from it are also signalling molecules that modulate growth and development, but the importance for signalling of endogenous changes in sugar levels is poorly understood. We report that reduced activity of cytosolic invertase, which converts sucrose to hexoses, leads to pronounced metabolic, growth and developmental defects in roots of Arabidopsis (Arabidopsis thaliana) seedlings. In addition to altered sugar and downstream metabolite levels, roots of cinv1 cinv2 mutants have reduced elongation rates, cell and meristem size, abnormal meristematic cell division patterns, and altered expression of thousands of genes of diverse functions. Provision of exogenous glucose to mutant roots repairs relatively few of the defects. The extensive transcriptional differences between mutant and wild-type roots have hallmarks of both high sucrose and low hexose signalling. We conclude that the mutant phenotype reflects both low carbon availability for metabolism and growth and complex sugar signals derived from elevated sucrose and depressed hexose levels in the cytosol of mutant roots. Such reciprocal changes in endogenous sucrose and hexose levels potentially provide rich information about sugar status that translates into flexible adjustments of growth and development.

Changes in sucrose metabolism in maize varieties with different cadmium sensitivities under cadmium stress

Sucrose metabolism contributes to the growth and development of plants and helps plants cope with abiotic stresses, including stress from Cd. Many of these processes are not well-defined, including the mechanism underlying the response of sucrose metabolism to Cd stress. In this study, we investigated how sucrose metabolism in maize varieties with low (FY9) and high (SY33) sensitivities to Cd changed in response to different levels of Cd (0 (control), 5, 10, and 20 mg L−1 Cd). The results showed that photosynthesis was impaired, and the biomass decreased, in both varieties of maize at different Cd concentrations. Cd inhibited the activities of sucrose phosphate synthase (SPS) and sucrose synthase (SS) (sucrose synthesis), and stimulated the activities of acid invertase (AI) and SS (sucrose hydrolysis). The total soluble sugar contents were higher in the Cd-treated seedlings than in the control. Also, Cd concentrations in the shoots were higher in SY33 than in FY9, and in the roots were lower in SY33 than in FY9. The decreases in the photosynthetic rate, synthesis of photosynthetic products, enzyme activity in sucrose synthesis direction, and increases in activity in hydrolysis direction were more obvious in SY33 (the sensitive variety) than in FY9 (the tolerant variety), and more photosynthetic products were converted into soluble sugar in SY33 than in FY9 as the Cd stress increased. The transcript levels of the sugar transporter genes also differed between the two varieties at different concentrations of Cd. These results suggest that sucrose metabolism may be a secondary response to Cd additions, and that the Cd-sensitive variety used more carbohydrates to defend against Cd stress rather than to support growth than the Cd-tolerant variety.