Hydrophobin signal sequence mediates efficient secretion of recombinant proteins in Pichia pastoris.

Pichia pastoris is an important eukaryotic organism for the expression, processing, and secretion of recombinant proteins. Here, the secretion of enhanced green fluorescent protein (EGFP) in P. pastoris by using three novel secretion signals originating from the HFBI and HFBII class 2 hydrophobins of Trichoderma reesei was investigated. EGFP was fused to the carboxyl terminus of hydrophobin secretion signals and expressed under the control of the constitutive GAP promoter. In every case, recombinant EGFP entered the secretory pathway of P. pastoris. SDS-polyacrylamide gel electrophoresis, Western blot analysis of the cells' supernatant, and fluorescence measurements on single-cell level via flow cytometry confirmed the efficient secretion of EGFP mediated by the novel secretion sequences. In conclusion, the data clearly show that the secretion sequences derived from HFBI and HFBII of T. reesei have the potential to achieve an efficient secretion of heterologous proteins in P. pastoris. Due to the small size of the hydrophobin-derived secretion signals, their coding sequence can be easily introduced to the gene of interest by PCR.

Increased product formation induced by a directed secondary substrate limitation in a batch Hansenula polymorpha culture.

By the use of directed limitations of secondary substrates, the metabolic flux should be deflected from biomass production to product formation. In order to study the impact of directed limitations caused by various secondary substrates on the growth and product formation of the methylotrophic yeast Hansenula polymorpha, the cultivation systems respiration activity monitoring system (RAMOS) and BioLector were used in parallel. While the RAMOS device allows the online monitoring of the oxygen transfer rate in shake flasks, the BioLector enables in microtiter plates the monitoring of scattered light and the fluorescence intensity of the green fluorescent protein (GFP). Secondary substrate limitations of phosphate, potassium, and magnesium were analyzed in batch fermentations. The sole carbon source was either 10 g/L glucose or 10 g/L glycerol. The expression of the GFP gene is controlled by the FMD promoter (formate dehydrogenase). In batch cultures with glucose as carbon source, a directed limitation of phosphate increased the GFP production 1.87-fold, compared to phosphate unlimited conditions. Under potassium-limited conditions with glycerol as sole carbon source, the GFP production was 1.41-fold higher compared to unlimited conditions. A limitation of the substrate magnesium resulted in a 1.22-fold increase GFP formation in the case of glycerol as carbon source.

Asymmetric division of Hansenula polymorpha reflected by a drop of light scatter intensity measured in batch microtiter plate cultivations at phosphate limitation.

Hansenula polymorpha RB11 pC10-FMD (P(FMD)- GFP) (FMD promoter gfp gene) was simultaneously cultivated in the Respiration Activity Monitoring System (RAMOS) and in the microtiter plate cultivation system "BioLector" under phosphate limitation. The light scatter signal of the BioLector, for the determination of the biomass concentration in the wells, shows a significant decrease with the onset of the phosphate limitation until a stationary level is reached. At lower initial phosphate concentration this effect is more pronounced and longer time is required until the stationary level of the scattered light is achieved. The oxygen transfer rate signal of the RAMOS and the light scatter signal of the BioLector correlate with respect to the points of time where the maxima and the stationary levels of the courses are reached. In order to understand the effect causing this light scatter behavior, the forward and side scatter properties were investigated off line by flow cytometry. The decay in the light scatter of the BioLector seems to correlate with the formation of two subpopulations of different scatter intensities detected by a flow cytometer. With ongoing cultivation the fraction of cells possessing higher light scattering properties decreases until only a population of lower light scattering properties exists. The rate of transition of the yeast from one subpopulation to the other appears to be correlated with the rate of decrease in the BioLector light scatter signal. The formation of the subpopulations may be caused by an increased asymmetry in the cell cycle due to phosphate limitation.