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

Background: Aspergillus niger is widely used for enzyme production and achievement of high enzyme production depends on the comprehensive understanding of cell's metabolic regulation mechanisms. Results: In 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. Conclusions: The 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 abstract An 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)OLD BT/Cell Systems EngineeringBT/Bioprocess Engineerin

Euler-Lagrange computational fluid dynamics for (bio)reactor scale down: An analysis of organism lifelines

The trajectories, referred to as lifelines, of individual microorganisms in an industrial scale fermentor under substrate limiting conditions were studied using an Euler-Lagrange computational fluid dynamics approach. The metabolic response to substrate concentration variations along these lifelines provides deep insight in the dynamic environment inside a large-scale fermentor, from the point of view of the microorganisms themselves. We present a novel methodology to evaluate this metabolic response, based on transitions between metabolic “regimes” that can provide a comprehensive statistical insight in the environmental fluctuations experienced by microorganisms inside an industrial bioreactor. These statistics provide the groundwork for the design of representative scale-down simulators, mimicking substrate variations experimentally. To focus on the methodology we use an industrial fermentation of Penicillium chrysogenum in a simplified representation, dealing with only glucose gradients, single-phase hydrodynamics, and assuming no limitation in oxygen supply, but reasonably capturing the relevant timescales. Nevertheless, the methodology provides useful insight in the relation between flow and component fluctuation timescales that are expected to hold in physically more thorough simulations. Microorganisms experience substrate fluctuations at timescales of seconds, in the order of magnitude of the global circulation time. Such rapid fluctuations should be replicated in truly industrially representative scale-down simulators.ChemE/Transport PhenomenaOLD BT/Cell Systems EngineeringBT/Bioprocess Engineerin

Multi-omics integrative analysis with genome-scale metabolic model simulation reveals global cellular adaptation of Aspergillus niger under industrial enzyme production condition

Oxygen limitation is regarded as a useful strategy to improve enzyme production by mycelial fungus like Aspergillus niger. However, the intracellular metabolic response of A. niger to oxygen limitation is still obscure. To address this, the metabolism of A. niger was studied using multi-omics integrated analysis based on the latest GEMs (genome-scale metabolic model), including metabolomics, fluxomics and transcriptomics. Upon sharp reduction of the oxygen supply, A. niger metabolism shifted to higher redox level status, as well as lower energy supply, down-regulation of genes for fatty acid synthesis and a rapid decrease of the specific growth rate. The gene expression of the glyoxylate bypass was activated, which was consistent with flux analysis using the A. niger GEMs iHL1210. The increasing flux of the glyoxylate bypass was assumed to reduce the NADH formation from TCA cycle and benefit maintenance of the cellular redox balance under hypoxic conditions. In addition, the relative fluxes of the EMP pathway were increased, which possibly relieved the energy demand for cell metabolism. The above multi-omics integrative analysis provided new insights on metabolic regulatory mechanisms of A. niger associated with enzyme production under oxygen-limited condition, which will benefit systematic design and optimization of the A. niger microbial cell factory.BT/Bioprocess Engineerin

Comparative performance of different scale-down simulators of substrate gradients in Penicillium chrysogenum cultures: the need of a biological systems response analysis

In a 54 m3 large-scale penicillin fermentor, the cells experience substrate gradient cycles at the timescales of global mixing time about 20–40 s. Here, we used an intermittent feeding regime (IFR) and a two-compartment reactor (TCR) to mimic these substrate gradients at laboratory-scale continuous cultures. The IFR was applied to simulate substrate dynamics experienced by the cells at full scale at timescales of tens of seconds to minutes (30 s, 3 min and 6 min), while the TCR was designed to simulate substrate gradients at an applied mean residence time ((Formula presented.)) of 6 min. A biological systems analysis of the response of an industrial high-yielding P. chrysogenum strain has been performed in these continuous cultures. Compared to an undisturbed continuous feeding regime in a single reactor, the penicillin productivity (qPenG) was reduced in all scale-down simulators. The dynamic metabolomics data indicated that in the IFRs, the cells accumulated high levels of the central metabolites during the feast phase to actively cope with external substrate deprivation during the famine phase. In contrast, in the TCR system, the storage pool (e.g. mannitol and arabitol) constituted a large contribution of carbon supply in the non-feed compartment. Further, transcript analysis revealed that all scale-down simulators gave different expression levels of the glucose/hexose transporter genes and the penicillin gene clusters. The results showed that qPenG did not correlate well with exposure to the substrate regimes (excess, limitation and starvation), but there was a clear inverse relation between qPenG and the intracellular glucose level.ChemE/Transport PhenomenaOLD BT/Cell Systems EngineeringBT/Bioprocess Engineerin