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Autolysis of Pichia pastoris induced by cold.

ABSTRACT: The production of recombinant biopharmaceutical proteins is a multi-billion dollar market. Protein recovery represents a major part of the production costs. Pichia pastoris is one of the microbial systems most used for the production of heterologous proteins. The use of a cold-induced promoter to express lytic enzymes in the yeast after the growth stage could reduce protein recovery costs. This study shows that a cold-shock can be applied to induce lysis of the yeast cells. A strain of P. pastoris was constructed in which the endogenous eng gene encoding a putative endo-?-1,3-glucanase was overexpressed using the cold-shock induced promoter of the cct? gene from Saccharomyces cerevisiae. In the transgenic P. pastoris, the expression of eng increased 3.6-fold after chilling the cells from 30 to 4 °C (cold-shock stage) followed by incubation for 6 h (eng expression stage). The culture was heated to 30 °C for 6 h (ENG synthesis stage) and kept at 37 °C for 24 h (lysis stage). After this procedure the cell morphology changed, spheroplasts were obtained and cellular lysis was observed. Thus, a clone of P. pastoris was obtained, which undergoes autolysis after a cold-shock.

SUBMITTER: Bartolo-Aguilar Y

PROVIDER: S-EPMC5429318 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Autolysis of Pichia pastoris induced by cold.

Bartolo-Aguilar Yaneth Y Dendooven Luc L Chávez-Cabrera Cipriano C Flores-Cotera Luis B LB Hidalgo-Lara María E ME Villa-Tanaca Lourdes L Marsch Rodolfo R

AMB Express 20170512 1

The production of recombinant biopharmaceutical proteins is a multi-billion dollar market. Protein recovery represents a major part of the production costs. Pichia pastoris is one of the microbial systems most used for the production of heterologous proteins. The use of a cold-induced promoter to express lytic enzymes in the yeast after the growth stage could reduce protein recovery costs. This study shows that a cold-shock can be applied to induce lysis of the yeast cells. A strain of P. pastor ...[more]

PMID: 28500590

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Project description:Over the past three decades, due to the universal application of Pichia yeast in the fermentation industry as well as the establishment of Pichia pastoris fermentation process for more than 30 years, the technology of the whole process has become very mature and has now reached a stagnated period of growth. However, studies and research conducted on the genomics of the classic fermentation process and the uncovering of the biological phenomena in the fermentation process from the point of view of high-throughput gene or protein is still in its early stages, and there is still insufficient data within this field. First, in collaboration with Agilent Company, we designed and prepared an expression microarray that could be used for the detection of Pichia pastoris transcriptomics. The transcriptomic changes in the five key technology steps (time points), during the fermentation process of Pichia pastoris would then be detected with the aid of an expression microarray. The five key steps of technology described above formed two important biological processes, namely, the limiting carbon source replacement and secondly, the fermentative production of exogenous proteins. The biological phenomena involved in these two processes were displayed and analyzed at the transcriptional level. In addition to this, with regard to the most important function in the fermentation process of Pichia pastoris, oxid-reduction, the metabolic drift process was analyzed and the important genes that might dominate the changes in the metabolic flux were discovered creatively by using the function tree method in this paper. This study was undertaken from the point of view of the transcriptome and the biological phenomena in the fermemntation process of Pichia pastoris. Both of which, were thoroughly explained during this study. The hope is for many more researchers to optimize the strain fermentation process, to produce proteins at the genetic level, as well as providing and obtaining new perspectives and detailed scientific data for the continued development within this field.

Project description:Pichia pastoris has emerged as an important alternative host for producing recombinant biopharmaceuticals, owing to its high cultivation density, low host cell protein burden, and the development of strains with humanized glycosylation. Despite its demonstrated utility, relatively little strain engineering has been performed to improve Pichia, due in part to the limited number and inconsistent frameworks of reported genomes and transcriptomes. Furthermore, the co-mingling of genomic, transcriptomic and fermentation data collected about Komagataella pastoris and Komagataella phaffii, the two strains co-branded as Pichia, has generated confusion about host performance for these genetically distinct species. Generation of comparative high-quality genomes and transcriptomes will enable meaningful comparisons between the organisms, and potentially inform distinct biotechnological utilies for each species.Here, we present a comprehensive and standardized comparative analysis of the genomic features of the three most commonly used strains comprising the tradename Pichia: K. pastoris wild-type, K. phaffii wild-type, and K. phaffii GS115. We used a combination of long-read (PacBio) and short-read (Illumina) sequencing technologies to achieve over 1000X coverage of each genome. Construction of individual genomes was then performed using as few as seven individual contigs to create gap-free assemblies. We found substantial syntenic rearrangements between the species and characterized a linear plasmid present in K. phaffii. Comparative analyses between K. phaffii genomes enabled the characterization of the mutational landscape of the GS115 strain. We identified and examined 35 non-synonomous coding mutations present in GS115, many of which are likely to impact strain performance. Additionally, we investigated transcriptomic profiles of gene expression for both species during cultivation on various carbon sources. We observed that the most highly transcribed genes in both organisms were consistently highly expressed in all three carbon sources examined. We also observed selective expression of certain genes in each carbon source, including many sequences not previously reported as promoters for expression of heterologous proteins in yeasts.Our studies establish a foundation for understanding critical relationships between genome structure, cultivation conditions and gene expression. The resources we report here will inform and facilitate rational, organism-wide strain engineering for improved utility as a host for protein production.

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Autolysis of Pichia pastoris induced by cold.

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Autolysis of Pichia pastoris induced by cold.

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