Dataset Information

A toolbox of endogenous and heterologous nuclear localization sequences for the methylotrophic yeast Pichia pastoris.

ABSTRACT: Nuclear localization sequences (NLSs) are required for the import of proteins in the nucleus of eukaryotes. However many proteins from bacteria or bacteriophages are used for basic studies in molecular biology, to generate synthetic genetic circuits or for genome editing applications. Prokaryotic recombinases, CRISPR-associated proteins such as Cas9 or bacterial and viral polymerases require efficient NLSs to function in eukaryotes. The yeast Pichia pastoris is a widely used expression platform for heterologous protein production, but molecular tools such as NLSs are limited. Here we have characterized a set of 10 NLSs for P. pastoris, including the first endogenous NLSs (derived from P. pastoris proteins) and commonly used heterologous NLSs. The NLSs were evaluated by fusing them in N- and C-terminal position to an enhanced green fluorescent protein showing pronounced differences in fluorescence levels and nuclear targeting. Thereby we provide a set of different NLSs that can be applied to optimize the nuclear import of heterologous proteins in P. pastoris, paving the way for the establishment of intricate synthetic biology applications.

SUBMITTER: Weninger A

PROVIDER: S-EPMC4629791 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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A toolbox of endogenous and heterologous nuclear localization sequences for the methylotrophic yeast Pichia pastoris.

Weninger Astrid A Glieder Anton A Vogl Thomas T

FEMS yeast research 20150906 7

Nuclear localization sequences (NLSs) are required for the import of proteins in the nucleus of eukaryotes. However many proteins from bacteria or bacteriophages are used for basic studies in molecular biology, to generate synthetic genetic circuits or for genome editing applications. Prokaryotic recombinases, CRISPR-associated proteins such as Cas9 or bacterial and viral polymerases require efficient NLSs to function in eukaryotes. The yeast Pichia pastoris is a widely used expression platform ...[more]

PMID: 26347503

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Project description:BackgroundPichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.ResultsA fully compartmentalized metabolic model of P. pastoris (iPP668), composed of 1,361 reactions and 1,177 metabolites, was reconstructed based on its genome annotation and biochemical information. The constraints-based flux analysis was then used to predict achievable growth rate which is consistent with the cellular phenotype of P. pastoris observed during chemostat experiments. Subsequent in silico analysis further explored the effect of various carbon sources on cell growth, revealing sorbitol as a promising candidate for culturing recombinant P. pastoris strains producing heterologous proteins. Interestingly, methanol consumption yields a high regeneration rate of reducing equivalents which is substantial for the synthesis of valuable pharmaceutical precursors. Hence, as a case study, we examined the applicability of P. pastoris system to whole-cell biotransformation and also identified relevant metabolic engineering targets that have been experimentally verified.ConclusionThe genome-scale metabolic model characterizes the cellular physiology of P. pastoris, thus allowing us to gain valuable insights into the metabolism of methylotrophic yeast and devise possible strategies for strain improvement through in silico simulations. This computational approach, combined with synthetic biology techniques, potentially forms a basis for rational analysis and design of P. pastoris metabolic network to enhance humanized glycoprotein production.

Dataset Information

A toolbox of endogenous and heterologous nuclear localization sequences for the methylotrophic yeast Pichia pastoris.

Publications

A toolbox of endogenous and heterologous nuclear localization sequences for the methylotrophic yeast Pichia pastoris.

Similar Datasets

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