Abstract
BackgroundYeast mating provides an efficient means for strain and library construction. However, biotechnological applications of mating in the methylotrophic yeast Pichia pastoris have been hampered because of concerns about strain stability of P. pastoris diploids. The aim of the study reported here is to investigate heterologous protein expression in diploid P. pastoris strains and to evaluate diploid strain stability using high cell density fermentation processes.ResultsBy using a monoclonal antibody as a target protein, we demonstrate that recombinant protein production in both wild-type and glycoengineered P. pastoris diploids is stable and efficient during a nutrient rich shake flask cultivation. When diploid strains were cultivated under bioreactor conditions, sporulation was observed. Nevertheless, both wild-type and glycoengineered P. pastoris diploids showed robust productivity and secreted recombinant antibody of high quality. Specifically, the yeast culture maintained a diploid state for 240 h post-induction phase while protein titer and N-linked glycosylation profiles were comparable to that of a haploid strain expressing the same antibody. As an application of mating, we also constructed an antibody display library and used mating to generate novel full-length antibody sequences.ConclusionsTo the best of our knowledge, this study reports for the first time a comprehensive characterization of recombinant protein expression and fermentation using diploid P. pastoris strains. Data presented here support the use of mating for various applications including strain consolidation, variable-region glycosylation antibody display library, and process optimization.
Highlights
Yeast mating provides an efficient means for strain and library construction
In order to understand the stability of diploids during methanol induction, plasmid pGLY10969 contains mCherry, a red fluorescent protein (RFP), and plasmid pGLY10970 includes yEGFP3, a green fluorescent protein (GFP), both regulated by the constitutive TEF promoter and the AOX1 transcription terminator
The main purpose of this study is to investigate utilization of diploid P. pastoris strains for heterologous protein expression and to understand diploid strain stability in high-productivity fermentation
Summary
Yeast mating provides an efficient means for strain and library construction. biotechnological applications of mating in the methylotrophic yeast Pichia pastoris have been hampered because of concerns about strain stability of P. pastoris diploids. The aim of the study reported here is to investigate heterologous protein expression in diploid P. pastoris strains and to evaluate diploid strain stability using high cell density fermentation processes. The methylotrophic yeast P. pastoris has become an increasingly popular host for recombinant protein expression in recent times. Being an obligate aerobe when fed with methanol, P. pastoris does not switch to anaerobic metabolism that would lead to toxic metabolite accumulation under oxygen limited condition. This makes it possible to run high cell density fermentations under dissolved oxygen controlled processes. Other benefits of the P. pastoris system include ease of genetic manipulation, stable expression, rapid cell growth, low-cost scalable fermentation processes and little to no risk of human pathogenic virus contamination. Fermentation titers at grams per liter scale have been reported for several target proteins including full-length antibodies [2,3,4,5,6]
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