In the present work, by performing chemostat experiments at 400 and 600 RPM, two typical power inputs representative of industrial penicillin fermentation (P/V, 1.00 kW/m3 in more remote zones and 3.83 kW/m3 in the vicinity of the impellers, respectively) were scaled-down to bench-scale bioreactors. It was found that at 400 RPM applied in prolonged glucose-limited chemostat cultures, the previously reported degeneration of penicillin production using an industrial Penicillium chrysogenum strain was virtually absent. To investigate this, the cellular response was studied at flux (stoichiometry), residual glucose, intracellular metabolite and transcript levels. At 600 RPM, 20% more cell lysis was observed and the increased degeneration of penicillin production was accompanied by a 22% larger ATP gap and an unexpected 20-fold decrease in the residual glucose concentration (Cs,out). At the same time, the biomass specific glucose consumption rate (qs) did not change but the intracellular glucose concentration was about sixfold higher, which indicates a change to a higher affinity glucose transporter at 600 RPM. In addition, power input differences cause differences in the diffusion rates of glucose and the calculated Batchelor diffusion length scale suggests the presence of a glucose diffusion layer at the glucose transporting parts of the hyphae, which was further substantiated by a simple proposed glucose diffusion-uptake model. By analysis of calculated mass action ratios (MARs) and energy consumption, it indicated that at 600 RPM glucose sensing and signal transduction in response to the low Cs,out appear to trigger a gluconeogenic type of metabolic flux rearrangement, a futile cycle through the pentose phosphate pathway (PPP) and a declining redox state of the cytosol. In support of the change in glucose transport and degeneration of penicillin production at 600 RPM, the transcript levels of the putative high-affinity glucose/hexose transporter genes Pc12g02880 and Pc06g01340 increased 3.5- and 3.3-fold, respectively, and those of the pcbC gene encoding isopenicillin N-synthetase (IPNS) were more than twofold lower in the time range of 100–200 hr of the chemostat cultures. Summarizing, changes at power input have unexpected effects on degeneration and glucose transport, and result in significant metabolic rearrangements. These findings are relevant for the industrial production of penicillin, and other fermentations with filamentous microorganisms.@en

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.@en

In Penicillium chrysogenum, it has been observed that turnover of storage carbohydrates (trehalose, mannitol, arabitol, erythritol and glycogen) resulting in an extra ATP expenditure might partly account for the reduced penicillin productivity under dynamic cultivation conditions. In this work, Penicillium chrysogenum mutants with altered trehalose metabolism were constructed using the Agrobacterium-mediated transformation method. It was observed that impaired trehalose biosynthesis did not result in growth arrest and change of glucose sensitivity to high glucose levels, but negatively influenced the sporulation. Compared with the original strain, in glucose-limited chemostat cultures, the biomass yield on glucose and energy efficiency were slightly enhanced; however, the penicillin productivity was significantly lowered in the trehalose mutant strains. Comparison with a high-yielding P. chrysogenum strain revealed that the original and mutant strains had a lower glucose uptake capacity but higher intracellular levels of free amino acids. Flux estimates through the central carbon metabolism showed distinctive difference in the upper part of the glycolysis and in the pentose phosphate pathway but comparable flux through the TCA cycle. Combining, the striking phenotypic effects observed in the trehalose mutants of P. chrysogenum indicated that trehalose metabolism plays an important role in metabolic regulation and is central to maintaining higher penicillin productivity under glucose-limited chemostat cultures.@en