Biomarkers allow detection of nutrient limitations and respective supplementation for elimination in Pichia pastoris fed-batch cultures

BACKGROUND Industrial processes for recombinant protein production challenge production hosts, such as the yeast Pichia pastoris, on multiple levels. During a common P. pastoris fed-batch process, cells experience strong adaptations to different metabolic states or suffer from environmental stresses due to high cell density cultivation. Additionally, recombinant protein production and nutrient limitations are challenging in these processes. RESULTS Pichia pastoris producing porcine carboxypeptidase B (CpB) was cultivated in glucose or methanol-limited fed-batch mode, and the cellular response was analyzed using microarrays. Thereby, strong transcriptional regulations in transport-, regulatory- and metabolic processes connected to sulfur, phosphorus and nitrogen metabolism became obvious. The induction of these genes was observed in both glucose- and methanol- limited fed batch cultivations, but were stronger in the latter condition. As the transcriptional pattern was indicative for nutrient limitations, we performed fed-batch cultivations where we added the respective nutrients and compared them to non-supplemented cultures regarding cell growth, productivity and expression levels of selected biomarker genes. In the non-supplemented reference cultures we observed a strong increase in transcript levels of up to 89-fold for phosphorus limitation marker genes in the late fed-batch phase. Transcript levels of sulfur limitation marker genes were up to 35-fold increased. By addition of (NH4)2SO4 or (NH4)2HPO4, respectively, we were able to suppress the transcriptional response of the marker genes to levels initially observed at the start of the fed batch. Additionally, supplementation had also a positive impact on biomass generation and recombinant protein production. Supplementation with (NH4)2SO4 led to 5% increase in biomass and 52% higher CpB activity in the supernatant, compared to the non-supplemented reference cultivations. In (NH4)2HPO4 supplemented cultures 9% higher biomass concentrations and 60% more CpB activity were reached. CONCLUSIONS Transcriptional analysis of P. pastoris fed-batch cultivations led to the identification of nutrient limitations in the later phases, and respective biomarker genes for indication of limitations. Supplementation of the cultivation media with those nutrients eliminated the limitations on the transcriptional level, and was also shown to enhance productivity of a recombinant protein. The biomarker genes are versatily applicable to media and process optimization approaches, where tailor-made solutions are envisioned.