Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks

Background - Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A2a adenosine receptor (hA2aR), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). Results - Functional hA2aR was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA2aR and GFP were still produced in the pre-induction phases. Both hA2aR and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. Conclusions - The production of recombinant hA2aR, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields

Investigation of the induced protein production in Aspergillus niger using the flourescent reporter protein S65TGFP

Das in Aspergillus niger produzierte Enzym Glucoamylase wird von der Zuckerindustrie häufig verwendet. Für die Genexpression in Aspergillus wird der A. niger-Glucoamylase-Promotor (glaA-Promotor) bevorzugt eingesetzt. In dieser Arbeit wurde die Regulation der glaA-Genexpression auf Transkriptionsebene mit Hilfe eines Modellsystems untersucht. Dabei wurde eine Mutationsform (S65TGFP) des fluoreszierenden Reporterproteins GFP ('green fluorescent protein') unter der Kontrolle des glaA-Promotors in A. niger produziert. Für diese Untersuchung wurde ein Screening-Verfahren entwickelt. Die Repression der glaA-kontrollierten Genexpression durch Xylose wurde nachgewiesen. Bei Wachstum auf Glucose und Kohlenstoffquellen mit a?1,4?glykosidisch gebundenen Glucoseeinheiten (Maltodextrin, Maltose, Maltotriose, Amylose) erfolgte eine Induktion der S65TGFP-Synthese. 2?Deoxyglucose erwies sich als nicht metabolisierbarer Induktor. Bei hohen Konzentrationen dieses Analogs der D?Glucose wurde eine Kohlenstoff-Repression festgestellt. Eine Proteinproduktion mit induzierter Produktbildung wurde mittels Fed-Batch-Kultivierungstechnik erreicht. Dabei wurde die S65TGFP-Produktion nach Wachstum auf Xylose (Batch-Phase) durch Zufütterung von Maltose induziert. Wachstumslimitierende Maltosezufütterung führte zu einer höheren Induktionseffizienz als Maltose-Überschuss-Zufütterung. Die S65TGFP-Produktion konnte mit Hilfe der in-situ-2D?Fluoreszenzspektroskopie in Echtzeit online verfolgt werden. Die spezifische S65TGFP-Konzentration war von der Pelletgröße abhängig. Mit Abnahme der Pelletgröße erhöhte sich die spezifische S65TGFP-Konzentration.The enzyme Glucoamylase, produced in Aspergillus niger, is frequently used in the sugar industry. The glucoamylase-promotor (glaA-promotor) of A. niger is favoured for gene-expression in Aspergillus. In this thesis the glaA-Genregulation on transcription level was studied, using a model system. A mutant form (S65TGFP) of the fluorescent reporter protein GFP (green fluorescent protein) was produced under control of the glaA-promotor. For this investigation a screening method was developed. Repression of the glaA-controlled gene-expression by Xylose was demonstrated. When A. niger was grown on glucose and carbon sources with a?1,4?linked glucose units (maltodextrin, maltose, maltotriose, amylose) synthesis of S65TGFP was induced. 2?Deoxyglucose acted as a non-metabolizable inducer. Carbon repression was demonstrated at high concentrations of this analogue of D?glucose. Protein production with induced product formation was achieved using fed-batch cultivation techniques. After growth on xylose (batch-phase), induction was accomplished by maltose feeding. The growth-limiting addition of maltose resulted in more efficient induction than excess maltose feeding. Online-monitoring of the production of S65TGFP in real time was demonstrated using in-situ-2D?fluorescence spectroscopy. Specific S65TGFP-concentration of pellets was shown to depend on pellet size. With decreasing pellet size, specific S65TGFP-concentration increased

In-depth analysis of the Aspergillus niger glucoamylase (glaA) promoter performance using high-throughput screening and controlled bioreactor cultivation techniques.

An in-depth characterization of the Aspergillus niger glucoamylase (glaA) promoter performance was carried out on defined medium employing multi-well high-throughput screening as well as controlled batch and fed-batch bioreactor culture techniques with GFP as a fluorescent reporter protein. A variety of metabolizable carbon substrates and non-metabolizable analogs were screened with regard to their effect on the glaA expression system. The results clearly demonstrate that only starch and its hydrolytic products, including glucose, act as inducers. However, induction of the glaA expression system through the monosaccharide glucose is significantly lower compared to starch and the higher molecular weight starch degradation products. All other 26 carbon substrates tested do not induce, or even, as in the case of the easily metabolizable monosaccharide xylose, repress glaA-promoter controlled gene expression in the presence of the inducing disaccharide maltose with an increase of repression strength by increasing xylose concentrations. The complex effect of glucose on glaA-promoter controlled expression was also analyzed using non-metabolizable glucose analogs, namely 5-thio-glucose and 2-deoxyglucose, which were identified as novel and potent inducers of the glaA expression system. The results show that the induction strength depends on the inducer concentration with a maximum at defined concentrations and lower induction or even repression at concentrations above. Moreover, controlled fed-batch cultivations using a high maltose feed rate with concomitant extracellular accumulation of glucose resulted in lower levels of the reporter protein compared to cultures with a low-maltose feed rate without extracellular glucose accumulation, thus supporting the conclusion that increasing the glucose concentration beyond a critical point reduces the induction strength or may even cause repression. This way, the speed of polymer hydrolysis, glucose uptake and intracellular breakdown can be fine-tuned for optimal fungal growth and the metabolic burden for glucoamylase synthesis can be limited adequately in response to nutrient availability

In situ multi-wavelength fluorescence spectroscopy as effective tool to simultaneously monitor spore germination, metabolic activity and quantitative protein production in recombinant Aspergillus niger fed-batch cultures.

The production of a mutant green fluorescent protein (S65TGFP), controlled by the maltose inducible glucoamylase promoter, was followed in situ in fed-batch cultures of recombinant Aspergillus niger using multi-wavelength fluorescence spectroscopy. Disturbance of quantitative product analysis by interfering fluorescence signals was resolved by using a set of defined combinations of excitation and emission wavelengths (lambda(ex)/lambda(em)). This technique resulted in excellent linearity between on-line signal and off-line determined S65TGFP concentrations. Spore germination was detectable in situ by monitoring the back scattered light intensity. Moreover, flavin-like fluorophores were identified as the dominating fungal host fluorophores. The time-dependent intensity of this fluorophore, potentially fungal flavin-containing oxidoreductase(s), did not correlate with the biomass concentration but correlated well with the fungal metabolic activity (e.g. respiratory activity). Other fluorophores commonly found in microbial cultures such NADH, pyridoxine and the aromatic amino acids, tryptophan, phenylalanine and tyrosine did not contribute significantly to the culture fluorescence of A. niger. Thus, multi-wavelength fluorescence spectroscopy has proven to be an effective tool for simultaneous on-line monitoring of the most relevant process variables in fungal cultures, e.g. spore germination, metabolic activity, and quantitative product formation