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.

Enzymatic microreactors for the determination of ethanol by an automatic sequential injection analysis system.

A sequential injection analysis system with two enzymatic microreactors for the determination of ethanol has been designed. Alcohol oxidase and horseradish peroxidase were separately immobilized on glass aminopropyl beads, and packed in 0.91-mL volume microreactors, working in line with the sequential injection analysis system.

Integrated biosensor systems for ethanol analysis.

Different integrated systems with a bi-enzymatic biosensor, working with two different methods for ethanol detection--flow injection analysis (FIA) or sequential injection analysis (SIA)--were developed and applied for ethanol extracted from gasohol mixtures, as well as for samples of alcoholic beverages and fermentation medium. A detection range of 0.05-1.

Hypoxic fed-batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins.

High cell density cultivation of Pichia pastoris has to cope with several technical limitations, most importantly the transfer of oxygen. By applying hypoxic conditions to chemostat cultivations of P. pastoris expressing an antibody Fab fragment under the GAP promoter, a 2.

A simple model-based control for Pichia pastoris allows a more efficient heterologous protein production bioprocess.

A predictive control algorithm coupled with a PI feedback controller has been satisfactorily implemented in the heterologous Rhizopus oryzae lipase production by Pichia pastoris methanol utilization slow (Mut(s)) phenotype. This control algorithm has allowed the study of the effect of methanol concentration, ranging from 0.5 to 1.

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.

Developing high cell density fed-batch cultivation strategies for heterologous protein production in Pichia pastoris using the nitrogen source-regulated FLD1 Promoter.

A Pichia pastoris strain expressing a Rhizopus oryzae lipase gene under the transcriptional control of the promoter from the P. pastoris formaldehyde dehydrogenase 1 gene (PFLD) was utilized to study the feasibility of this expression system for recombinant protein production using methanol-free fed-batch high cell density cultivations. We have developed a simple and reliable fed-batch strategy using the PFLD system based on the use of methylamine and sorbitol as nitrogen and carbon sources, respectively, for the induction phase.

Combined effect of the methanol utilization (Mut) phenotype and gene dosage on recombinant protein production in Pichia pastoris fed-batch cultures.

An important number of heterologous proteins have been produced in the methylotrophic yeast Pichia pastoris using the alcohol oxidase promoter. Two factors that drastically influence protein production and cultivation process development in this system are gene dosage and methanol assimilation capacity of the host strain (Mut phenotype). Using a battery of four strains which secrete a Rhizopus oryzae lipase (ROL), the combined effects of gene dosage and Mut phenotype on recombinant protein production in Pichia pastoris was studied in fed-batch cultures.