Abstract
BackgroundPichia pastoris is widely used as a production platform for heterologous proteins and model organism for organelle proliferation. Without a published genome sequence available, strain and process development relied mainly on analogies to other, well studied yeasts like Saccharomyces cerevisiae.ResultsTo investigate specific features of growth and protein secretion, we have sequenced the 9.4 Mb genome of the type strain DSMZ 70382 and analyzed the secretome and the sugar transporters. The computationally predicted secretome consists of 88 ORFs. When grown on glucose, only 20 proteins were actually secreted at detectable levels. These data highlight one major feature of P. pastoris, namely the low contamination of heterologous proteins with host cell protein, when applying glucose based expression systems. Putative sugar transporters were identified and compared to those of related yeast species. The genome comprises 2 homologs to S. cerevisiae low affinity transporters and 2 to high affinity transporters of other Crabtree negative yeasts. Contrary to other yeasts, P. pastoris possesses 4 H+/glycerol transporters.ConclusionThis work highlights significant advantages of using the P. pastoris system with glucose based expression and fermentation strategies. As only few proteins and no proteases are actually secreted on glucose, it becomes evident that cell lysis is the relevant cause of proteolytic degradation of secreted proteins. The endowment with hexose transporters, dominantly of the high affinity type, limits glucose uptake rates and thus overflow metabolism as observed in S. cerevisiae. The presence of 4 genes for glycerol transporters explains the high specific growth rates on this substrate and underlines the suitability of a glycerol/glucose based fermentation strategy. Furthermore, we present an open access web based genome browser .
Highlights
Pichia pastoris is widely used as a production platform for heterologous proteins and model organism for organelle proliferation
P. pastoris has recently been reclassified into a new genus, Komagataella [9], and split into three species, K. pastoris, K. phaffii, and K. pseudopastoris [10]
In order to provide a common information basis across the different strains, we have performed this work with the type strain (DSMZ 70382) of the type species K. pastoris, which is the reference strain for all the available P. pastoris strains
Summary
Pichia pastoris is widely used as a production platform for heterologous proteins and model organism for organelle proliferation. While protein production is the major application of P. pastoris, production of metabolites has come into research focus recently too [5,6] Apart from these biotechnological applications, it is widely used as a model for peroxisome [7] and secretory organelle research [8]. The strains GS115 and X-33 are K. phaffii, while the SMD series of protease deficient strains (most popular SMD1168) is classified into the type species, K. pastoris Apart from these strains which have been made available by Invitrogen, research labs and industry use different other strains belonging to either of these two species, and no trend towards a superior expression level of one of the two species has been observed. DSMZ 70382 was isolated from tree exudate, in this case from the chestnut tree
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