Simultaneous utilization of glucose and gluconate in Penicillium chrysogenum during overflow metabolism.

The filamentous fungus Penicillium chrysogenum is one of the most important production organism for ß-lactam antibiotics, especially penicillin. A specific feature of P. chrysogenum is the formation of gluconate as the primary overflow metabolite under non-limiting growth on glucose. Gluconate can be formed extracellularly by the enzyme glucose oxidase (GOD) that shows high activities under glucose excess conditions. Currently, it is assumed that under these conditions glucose is the preferred carbon substrate for P. chrysogenum and gluconate consumption first starts after glucose becomes limiting. Here, we specifically address this hypothesis by combining batch cultivation experiments on defined glucose media, time-dependent GOD activity measurements, and (13)C-tracer studies. Our data prove that both substrates are metabolized simultaneously independent from the actual glucose concentration and therefore suggest that no distinct mechanism of carbon catabolite repression exists for gluconate in P. chrysogenum. Moreover, gluconate consumption does not interfere with penicillin V production by repression of the penicillin genes. Finally, by following a model-driven approach the specific uptake rates for glucose and gluconate were quantified and found to be significantly higher for gluconate. In summary, our results show that P. chrysogenum metabolizes gluconate directly and at high rates making it an interesting alternative carbon source for production purposes.

Quantitative quenching evaluation and direct intracellular metabolite analysis in Penicillium chrysogenum.

Sustained progress in metabolic engineering methodologies has stimulated new efforts toward optimizing fungal production strains such as through metabolite analysis of Penicillium chrysogenum industrial-scale processes. Accurate intracellular metabolite quantification requires sampling procedures that rapidly stop metabolism (quenching) and avoid metabolite loss via the cell membrane (leakage). When sampling protocols are validated, the quenching efficiency is generally not quantitatively assessed. For fungal metabolomics, quantitative biomass separation using centrifugation is a further challenge. In this study, P. chrysogenum intracellular metabolites were quantified directly from biomass extracts using automated sampling and fast filtration. A master/slave bioreactor concept was applied to provide industrial production conditions. Metabolic activity during sampling was monitored by 13C tracing. Enzyme activities were efficiently stopped and metabolite leakage was absent. This work provides a reliable method for P. chrysogenum metabolomics and will be an essential base for metabolic engineering of industrial processes.