Statistically Designed Medium Reveals Interactions between Metabolism and Genetic Information Processing for Production of Stable Human Serum Albumin in Pichia pastoris.

Maity Maity,Mishra Mishra

doi:10.3390/biom9100568

Abstract

Human serum albumin (HSA), sourced from human serum, has been an important therapeutic protein for several decades. Pichia pastoris is strongly considered as an expression platform, but proteolytic degradation of recombinant HSA in the culture filtrate remains a major bottleneck for use of this system. In this study, we have reported the development of a medium that minimized proteolytic degradation across different copy number constructs. A synthetic codon-optimized copy of HSA was cloned downstream of α–factor secretory signal sequence and expressed in P. pastoris under the control of Alcohol oxidase 1 promoter. A two-copy expression cassette was also prepared. Culture conditions and medium components were identified and optimized using statistical tools to develop a medium that supported stable production of HSA. Comparative analysis of transcriptome data obtained by cultivation on optimized and unoptimized medium indicated upregulation of genes involved in methanol metabolism, alternate nitrogen assimilation, and DNA transcription, whereas enzymes of translation and secretion were downregulated. Several new genes were identified that could serve as possible targets for strain engineering of this yeast.

Highlights

Since the 1970s, an exponential rise in commercial production of pharmaceuticals has led to the development of several biotechnology industries
Experiments based on statistical design were chosen to identify factors affecting extracellular production of stable Human serum albumin (HSA) in the culture supernatant of P. pastoris
An analysis of 16 upregulated and 15 downregulated genes under two cultivation conditions was performed and the genes were mapped to pathways belonging to four categories

Summary

Introduction

Since the 1970s, an exponential rise in commercial production of pharmaceuticals has led to the development of several biotechnology industries. The methylotrophic yeast Pichia pastoris is considered an efficient expression platform for production of human therapeutics [2,3]. In spite of a large body of information available on the genetics of the yeast, little is known about how external fermentation conditions affect cell physiology and the network of metabolism and genetic information processing. Rapid methanol metabolism is accompanied by the accumulation of formaldehyde, leading to cellular toxicity. This can be partially alleviated by combining a second sugar, such as lactose [4], sorbitol [5], or glycerol [6] in the production phase, the regulation of methanol feed remains critical in Pichia fermentation. Temperature is considered to be important, as high temperature leads to accumulation of improperly folded proteins triggering stress pathways that lead to cell death [7]

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