Dataset Information

In Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response.

ABSTRACT: Protein production in yeasts is related to the specific growth rate μ. To elucidate on this correlation, we studied the transcriptome of Pichia pastoris at different specific growth rates by cultivating a strain secreting human serum albumin at μ = 0.015 to 0.15 h(-1) in glucose-limited chemostats. Genome-wide regulation revealed that translation-related as well as mitochondrial genes were upregulated with increasing μ, while autophagy and other proteolytic processes, carbon source-responsive genes and other targets of the TOR pathway as well as many transcriptional regulators were downregulated at higher μ. Mating and sporulation genes were most active at intermediate μ of 0.05 and 0.075 h(-1) . At very slow growth (μ = 0.015 h(-1) ) gene regulation differs significantly, affecting many transporters and glucose sensing. Analysis of a subset of genes related to protein folding and secretion reveals that unfolded protein response targets such as translocation, endoplasmic reticulum genes, and cytosolic chaperones are upregulated with increasing growth rate while proteolytic degradation of secretory proteins is downregulated. We conclude that a high μ positively affects specific protein secretion rates by acting on multiple cellular processes.

SUBMITTER: Rebnegger C

PROVIDER: S-EPMC4162992 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

In Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response.

Rebnegger Corinna C Graf Alexandra B AB Valli Minoska M Steiger Matthias G MG Gasser Brigitte B Maurer Michael M Mattanovich Diethard D

Biotechnology journal 20140114 4

Protein production in yeasts is related to the specific growth rate μ. To elucidate on this correlation, we studied the transcriptome of Pichia pastoris at different specific growth rates by cultivating a strain secreting human serum albumin at μ = 0.015 to 0.15 h(-1) in glucose-limited chemostats. Genome-wide regulation revealed that translation-related as well as mitochondrial genes were upregulated with increasing μ, while autophagy and other proteolytic processes, carbon source-responsive ge ...[more]

PMID: 24323948

Similar Datasets

Project description:Pichia pastoris (Komagataella phaffii) is widely used for industrial production of heterologous proteins due to high secretory capabilities but selection of highly productive engineered strains remains a limiting step. Despite availability of a comprehensive molecular toolbox for construct design and gene integration, there is high clonal variability among transformants due to frequent multi-copy and off-target random integration. Therefore, functional screening of several hundreds of transformant clones is essential to identify the best protein production strains. Screening methods are commonly based on deep-well plate cultures with analysis by immunoblotting or enzyme activity assays of post-induction samples, and each heterologous protein produced may require development of bespoke assays with multiple sample processing steps. In this work, we developed a generic system based on a P. pastoris strain that uses a protein-based biosensor to identify highly productive protein secretion clones from a heterogeneous set of transformants. The biosensor uses a split green fluorescent protein where the large GFP fragment (GFP1-10) is fused to a sequence-specific protease from Tobacco Etch Virus (TEV) and is targeted to the endoplasmic reticulum. Recombinant proteins targeted for secretion are tagged with the small fragment of the split GFP (GFP11). Recombinant protein production can be measured by monitoring GFP fluorescence, which is dependent on interaction between the large and small GFP fragments. The reconstituted GFP is cleaved from the target protein by TEV protease, allowing for secretion of the untagged protein of interest and intracellular retention of the mature GFP. We demonstrate this technology with four recombinant proteins (phytase, laccase, β-casein and β-lactoglobulin) and show that the biosensor directly reports protein production levels that correlate with traditional assays. Our results confirm that the split GFP biosensor can be used for facile, generic, and rapid screening of P. pastoris clones to identify those with the highest production levels.

Project description:To take full advantage of recombinant Pichia pastoris (Komagataella phaffii) as a production system for heterologous proteins, the complex protein secretory process should be understood and optimised by circumventing bottlenecks. Typically, little or no attention has been paid to the fate of newly synthesised protein inside the cell, or its passage through the secretory pathway, and only the secreted product is measured. However, the system's productivity (i.e. specific production rate qp), includes productivity of secreted (qp,extra) plus intracellularly accumulated (qp,intra) protein. In bioreactor cultivations with P. pastoris producing penicillin G acylase, we studied the dynamics of product formation, i.e. both the specific product secretion (qp,extra) and product retention (qp,intra) as functions of time, as well as the kinetics, i.e. productivity in relation to specific growth rate (μ). Within the time course, we distinguished (I) an initial phase with constant productivities, where the majority of product accumulated inside the cells, and qp,extra, which depended on μ in a bell-shaped manner; (II) a transition phase, in which intracellular product accumulation reached a maximum and productivities (intracellular, extracellular, overall) were changing; (III) a new phase with constant productivities, where secretion prevailed over intracellular accumulation, qp,extra was linearly related to μ and was up to three times higher than in initial phase (I), while qp,intra decreased 4-6-fold. We show that stress caused by heterologous protein production induces cellular imbalance leading to a secretory bottleneck that ultimately reaches equilibrium. This understanding may help to develop cultivation strategies for improving protein secretion from P. pastoris.Key Points• A novel concept for industrial bioprocess development.• A Relationship between biomass growth and product formation in P. pastoris.• A Three (3) phases of protein production/secretion controlled by the AOX1-promoter.• A Proof of concept in production of industrially relevant penicillin G acylase.

Dataset Information

In Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response.

Publications

In Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets