Oriol Cos

Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail.

Engineering of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated FLD1 promoter

The yeast Pichia pastoris has been previously used for extracellular expression of a Rhizopus oryzae lipase (Rol). However, limitations in Rol folding and secretion through the cell wall became apparent when producing it in fed-batch cultivations. In this study, we have investigated the effect of combining two cell engineering strategies to alleviate putative bottlenecks in Rol secretion, namely the constitutive expression of the induced form of the Saccharomyces cerevisiae unfolded protein response transcriptional factor Hac1 and the deletion of the GAS1 gene encoding beta-1,3-glucanosyltransglycosylase, GPI-anchored to the outer leaflet of the plasma membrane, playing a key role in yeast cell wall assembly. The performance of these engineered Rol-producing strains has been compared in fed-batch cultivations set at a low specific growth rate of about 0.005 h-(1). It was found that Rol overexpression in a P. pastoris strain expressing constitutively the induced form of S. cerevisiae Hac1 and the deletion of GAS1 resulted in about a 3-fold and 4-fold increase in the overall process specific productivity, respectively, whereas the double mutant HAC1/deltagas1 strain yielded about a 7-fold increase. Overall, these results reflect the multiplicity of physiological bottlenecks at different levels/steps throughout the Rol synthesis, secretion and excretion processes in P. pastoris.

Improving the monitoring of methanol concentration during high cell density fermentation of Pichia pastoris.

The Pichia pastoris expression system is widely used for the production of recombinant proteins. A simple and efficient experimental set-up allowing on-line monitoring of the methanol concentration during the fermentation of P. pastorisbased on the detection of the methanol vapor concentration in the exhaust air from fermenter by a tin dioxide (SnO2) semiconductor sensor is described. An experimental procedure to allow precise calibration of the system and to reduce methanol sensors interferences (>95% reduction) are also presented and discussed. Accuracy and measurement error were estimated about 0.05 g ⋅ l−1 and 6%, respectively. The efficient monitoring of methanol will help to advanced control of recombinant protein production and process optimization.

Colorimetric method for the determination of ethanol by Flow Injection Analysis

An automated Flow Injection Analysis system using stop-flow technique for quantifying ethanol based in a colorimetric detection method was developed. The system permitted analysis in a linear range of 0.05–1 g ethanol l−1 without external dilution, a sampling frequency of 15 analyses per hour, and a relative standard deviation of 3.5%. A dilution line was implemented in the FIA system permitting the extension of the linear range to 0.5–5.2 g ethanol l−1 maintaining the same sampling frequency and standard deviation. The system was applied to measure ethanol concentrations present in samples of an alcoholic fermentation and the results showed no significant difference with other analytical procedures (GC).

Substrate feeding strategies in Pichia pastoris fed-batch cultivation processes: Analysis of key parameters influencing recombinant protein production

has been studied in fed-batch bioprocesses witha manual (off-line) methanol concentration control [4].These studies demonstrated that variations of the residualmethanol concentration influence drastically in the spe-cific consumption and production rates. To avoid thisproblem a predictive control algorithm coupled with a PIfeedback controller has been satisfactorily implemented[5].This set-up has allowed for further analysis of several keyparameters influencing heterologous protein productionin

On-line monitoring of lipolytic activity by sequential injection analysis

An automated sequential injection analysis using stop-flow technique for the on-line determination of lipolytic activity has been developed. It is based on a colorimetric method using a chromogenic substrate, 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin)-ester. The system permits a linear range analysis between 5–100 lipolytic activity units ml−1, without external dilution of the sample, a sampling frequency of 5 samples per hour and a relative standard deviation (RSD) of 5%. The analyser has been used for the on-line monitoring of Candida rugosa fed-batch fermentation with excellent performance, regarding its reliability and reproducibility.

Analysis of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated formaldehyde dehydrogenase promoter (PFLD1)

Background Methanol-free high cell density fed-batch cultivation strategies for the P. pastoris expression system have been recently developed by expressing a Rhizopus oryzae lipase (ROL) under the transcriptional control of the PFLD1 [1]. These cultivation strategies were based on the use of sorbitol and methylamine as carbon and nitrogen source, respectively, during the induction phase of the cultivation process. Fed-batch fermentations were performed at three different specific growth rates and showed that productivities were strongly correlated with this parameter (i.e. with the cells physiological state). Moreover, intracellular active product accumulation and a decrease in the specific product secretion rate were observed along the induction phase of the fermentation process. These results suggested the presence of a bottleneck(s) throughout the synthesis and secretion process of the heterologous lipase.