During glucose-limited growth, a substantial input of adenosine triphosphate (ATP) is required for the production of β-lactams by the filamentous fungus Penicillium chrysogenum. Formate dehydrogenase has been confirmed in P. chrysogenum for formate oxidation allowing an extra supply of ATP, and coassimilation of glucose and formate has the potential to increase penicillin production and biomass yield. In this study, the steady-state metabolite levels and fluxes in response to cofeeding of formate as an auxiliary substrate in glucose-limited chemostat cultures at the dilution rates (D) of both 0.03 h⁻¹ and 0.05 h⁻¹ are determined to evaluate the quantitative impact on the physiology of a high-yielding P. chrysogenum strain. It is observed that an equimolar addition of formate is conducive to an increase in both biomass yield and penicillin production at D = 0.03 h⁻¹, while this is not the case at D = 0.05 h⁻¹. In addition, a higher cytosolic redox status (NADH/NAD⁺), a higher intracellular glucose level, and lower penicillin productivity are only observed upon formate addition at D = 0.05 h⁻¹, which are virtually absent at D = 0.03 h⁻¹. In conclusion, the results demonstrate that the effect of formate as an auxiliary substrate on penicillin productivity in the glucose-limited chemostat cultivations of P. chrysogenum is not only dependent on the formate/glucose ratio as published before but also on the specific growth rate. The results also imply that the overall process productivity and quality regarding the use of formate should be further explored in an actual industrial-scale scenario.

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.