The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses

Ali Kazemi Seresht,Lisbeth Olsson,Eva Akke Palmqvist

doi:10.1186/1475-2859-10-104

Abstract

BackgroundThe adaptation of unicellular organisms like Saccharomyces cerevisiae to alternating nutrient availability is of great fundamental and applied interest, as understanding how eukaryotic cells respond to variations in their nutrient supply has implications spanning from physiological insights to biotechnological applications.ResultsThe impact of a step-wise restricted supply of phosphate on the physiological state of S. cerevisiae cells producing human Insulin was studied. The focus was to determine the changes within the global gene expression of cells being cultured to an industrially relevant high cell density of 33 g/l cell dry weight and under six distinct phosphate concentrations, ranging from 33 mM (unlimited) to 2.6 mM (limited). An increased flux through the secretory pathway, being induced by the PHO circuit during low Pi supplementation, proved to enhance the secretory production of the heterologous protein. The re-distribution of the carbon flux from biomass formation towards increased glycerol production under low phosphate led to increased transcript levels of the insulin gene, which was under the regulation of the TPI1 promoter.ConclusionsOur study underlines the dynamic character of adaptive responses of cells towards a change in their nutrient access. The gradual decrease of the phosphate supply resulted in a step-wise modulated phenotypic response, thereby alternating the specific productivity and the secretory flux. Our work emphasizes the importance of reduced phosphate supply for improved secretory production of heterologous proteins.

Highlights

The adaptation of unicellular organisms like Saccharomyces cerevisiae to alternating nutrient availability is of great fundamental and applied interest, as understanding how eukaryotic cells respond to variations in their nutrient supply has implications spanning from physiological insights to biotechnological applications
Initially, we investigated the impact of a reduced phosphate supply on the metabolism and, more importantly, on the Insulin Analogue Precursor (IAP) productivity
The PHO5 promoter has been a preferred regulator of heterologous protein production, as cells which have been grown under phosphate limitation over-express this acid phosphatase gene and the heterologous protein that has been put under the inducible PHO5 promoter [22,23]

Summary

Introduction

The adaptation of unicellular organisms like Saccharomyces cerevisiae to alternating nutrient availability is of great fundamental and applied interest, as understanding how eukaryotic cells respond to variations in their nutrient supply has implications spanning from physiological insights to biotechnological applications. Despite the recent developments within the field of metabolic engineering and synthetic biology, which mostly target the production of metabolites like organic acids [3] and the reinforced alternatives to former petrochemical-based compounds (reviewed by [4] and [5]), only little novel engineering has been achieved in yeast with respect to its secretory abilities. The secretory machinery of eukaryotes like S. cerevisiae, embodying its severe quality control abilities and the performance of complex posttranslational modifications, is still providing a partly undiscovered and fruitful ground for biotechnological progress with respect to both quantitatively and qualitatively enhanced production of APIs. In particular, the popular metabolic engineering philosophy of channeling an increased flux towards a given metabolic pathway turns out to be more challenging when it comes to increasing the secretory flux of the cells, as engineering of yeast protein factories still remains on the level of chaperone and ER resident folding catalysts (reviewed by [8])

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