Integrated isotope-assisted metabolomics and 13C metabolic flux analysis reveals metabolic flux redistribution for high glucoamylase production by Aspergillus niger

Abstract

BackgroundAspergillus niger is widely used for enzyme production and achievement of high enzyme production depends on the comprehensive understanding of cell’s metabolic regulation mechanisms.ResultsIn this paper, we investigate the metabolic differences and regulation mechanisms between a high glucoamylase-producing strain A. niger DS03043 and its wild-type parent strain A. niger CBS513.88 via an integrated isotope-assisted metabolomics and 13C metabolic flux analysis approach. We found that A. niger DS03043 had higher cell growth, glucose uptake, and glucoamylase production rates but lower oxalic acid and citric acid secretion rates. In response to above phenotype changes, A. niger DS03043 was characterized by an increased carbon flux directed to the oxidative pentose phosphate pathway in contrast to reduced flux through TCA cycle, which were confirmed by consistent changes in pool sizes of metabolites. A higher ratio of ATP over AMP in the high producing strain might contribute to the increase in the PP pathway flux as glucosephosphate isomerase was inhibited at higher ATP concentrations. A. niger CBS513.88, however, was in a higher redox state due to the imbalance of NADH regeneration and consumption, resulting in the secretion of oxalic acid and citric acid, as well as the accumulation of intracellular OAA and PEP, which may in turn result in the decrease in the glucose uptake rate.ConclusionsThe application of integrated metabolomics and 13C metabolic flux analysis highlights the regulation mechanisms of energy and redox metabolism on flux redistribution in A. niger. Graphical abstractAn integrated isotope-assisted metabolomics and 13C metabolic flux analysis was was firstly systematically performed in A. niger. In response to enzyme production, the metabolic flux in A. niger DS03043 (high-producing) was redistributed, characterized by an increased carbon flux directed to the oxidative pentose phosphate pathway as well as an increased pool size of pentose. The consistency in 13C metabolic flux analysis and metabolites quantification indicated that an imbalance of NADH formation and consumption led to the accumulation and secretion of organic acids in A. niger CBS513.88 (wild-type)Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0329-y) contains supplementary material, which is available to authorized users.

Assessment of Metabolism of the Eastern Oyster and Eastern Elliptio Using NMR Spectroscopy

“…Experimental conditions to acquire 13 C CSI data are described in a previously published paper with the exception that no slice selection gradient was applied. 8 Since the main purpose of the 13 C imaging was to identify the spatial distribution of infused 13 C compounds, no slice selection was required and the resultant 13 C image was a projection image. Figure 1 shows the results of an MR imaging study of an oyster.…”

“…6 In short, Eastern oysters (Crassostrea virginica) (50-70 g) were collected from Taylor Creek near Beaufort, NC, USA, and freshwater mussels (Elliptio complanata) were collected from the inland creek near Beaufort, NC, USA. After fasting for a day, oysters were transferred to a beaker containing 200 mL of water and the appropriate 13 C labeled nutrients (5.5 mM 13 C-glucose or 2.7 mM 2-13 C-glycine, Cambridge Isotope Lab., Andover, MA, USA) for 48 h prior to dissection. Aeration was provided with a 1/4 inch diameter Tygon (Aqueon Products, Franklin, WI, USA) tube inserted into each beaker bubbling room air lightly to avoid startling the oyster into closing.…”

“…9,16,17 The 14.1 T Varian Inova spectrometer equipped with a 5 mm broadband NMR probe was used to acquire 13 C NMR spectra at 25 °C. The pulse and acquire sequence was used with WALTZ decoupling only during acquisition, a sweep width 32,000 Hz and 64,000 complex data points, a 2 second relaxation time, and the number of transients was 640.…”

“…9,15 Hence, glucose was chosen to probe glycolysis and the Krebs cycle, while glycine was chosen to probe betaine, which is trimethylglycine, a catabolic product of glycine. 14 Therefore, the major goal of this study was to establish baseline distributions of metabolites derived from U-13 C-glucose and 2-13 C-glycine over oyster tissues by using 13 C NMR measurement and to demonstrate the feasibility of using NMR for that purpose. In addition, we also performed the 1 H NMR measurements on the freshwater bivalve Eastern Elliptio (Elliptio complanata) which is not subject to a daily tidal cycle to measure the effect of 3 days fasting stress on metabolites.…”

“…The use of these techniques are possible since the background signal due to the natural abundance of 13 C is very small or negligible in most cases. 13 Here we have applied high-resolution 1 H NMR methods to fresh water bivalve Eastern Elliptio to investigate the variation in the metabolic profile of different organ groups after 3 days of fasting stress and 13 C NMR methods to Eastern oyster (Crassostrea virginica) to investigate the variation in the metabolic profile of different organ groups after infusing metabolically important NMR detectable stable isotopes, uniformly-labeled (U-13 C)-glucose or 2-13 C-glycine. 2,14 In a previous 1 H NMR metabolomic study, we have shown that betaine was the most abundant metabolite in all oyster tissues.…”