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Decreased expression of LRA4, a key gene involved in rhamnose metabolism, caused up-regulated expression of the genes in this pathway and autophagy in Pichia pastoris.

ABSTRACT: In a previous study, we developed Pichia pastoris GS115m, an engineered strain with decreased expression of one key gene, LRA4, in rhamnose metabolism. P. pastoris GS115m/LacB was subsequently constructed via introducing a ?-galactosidase gene, LacB, under the control of rhamnose-inducible PLRA3 into P. pastoris GS115m. P. pastoris GS115m/LacB greatly improved recombinant protein production relative to the parental strain (P. pastoris GS115/LacB). In the present study, transcriptomes of P. pastoris GS115m/LacB and P. pastoris GS115/LacB grown in YPR medium were analyzed. P. pastoris GS115m/LacB was found to suffer from the mild carbon source starvation. To attenuate the starvation stress, P. pastoris GS115m/LacB attempted to enhance rhamnose metabolism by elevating the transcription levels of rhamnose-utilization genes LRA1-3 and RhaR. The transcription level of LacB under the control of PLRA3 thereby increased, resulting in the improved production of recombinant protein in P. pastoris GS115m/LacB. It was also revealed that P. pastoris GS115m/LacB cells coped with carbon starvation mostly via autophagy.

SUBMITTER: Jiao J

PROVIDER: S-EPMC7042458 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Decreased expression of LRA4, a key gene involved in rhamnose metabolism, caused up-regulated expression of the genes in this pathway and autophagy in Pichia pastoris.

Jiao Jian J Wang Shuai S Tian Hui H Xu Xinxin X Zhang Yuhong Y Liu Bo B Zhang Wei W

AMB Express 20200225 1

In a previous study, we developed Pichia pastoris GS115m, an engineered strain with decreased expression of one key gene, LRA4, in rhamnose metabolism. P. pastoris GS115m/LacB was subsequently constructed via introducing a β-galactosidase gene, LacB, under the control of rhamnose-inducible P<sub>LRA3</sub> into P. pastoris GS115m. P. pastoris GS115m/LacB greatly improved recombinant protein production relative to the parental strain (P. pastoris GS115/LacB). In the present study, transcriptomes ...[more]

PMID: 32100129

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Project description:BackgroundInducible high-level expression is favoured for recombinant protein production in Pichia pastoris. Therefore, novel regulated promoters are desired, ideally repressing heterologous gene expression during initial growth and enabling it in the production phase. In a typical large scale fed-batch culture repression is desired during the batch phase where cells grow on a surplus of e.g. glycerol, while heterologous gene expression should be active in the feed phase under carbon (e.g. glucose) limitation.ResultsDNA microarray analysis of P. pastoris wild type cells growing in glycerol-based batch and glucose-based fed batch was used for the identification of genes with both, strong repression on glycerol and high-level expression in the feed phase. Six novel glucose-limit inducible promoters were successfully applied to express the intracellular reporter eGFP. The highest expression levels together with strong repression in pre-culture were achieved with the novel promoters P(G1) and P(G6). Human serum albumin (HSA) was used to characterize the promoters with an industrially relevant secreted protein. A P(G1) clone with two gene copies reached about 230% of the biomass specific HSA titer in glucose-based fed batch fermentation compared to a P(GAP) clone with identical gene copy number, while P(G6) only achieved 39%. Two clones each carrying eleven gene copies, expressing HSA under control of P(G1) and P(G6) respectively were generated by post-transformational vector amplification. They produced about 1.0 and 0.7 g L(-1) HSA respectively in equal fed batch processes. The suitability in production processes was also verified with HyHEL antibody Fab fragment for P(G1) and with porcine carboxypeptidase B for P(G6). Moreover, the molecular function of the gene under the control of P(G1) was determined to encode a high-affinity glucose transporter and named GTH1.ConclusionsA set of novel regulated promoters, enabling induction without methanol, was successfully identified by using DNA microarrays and shown to be suitable for high level expression of recombinant proteins in glucose-based protein production processes.

Project description:BACKGROUND: The effect of osmolarity on cellular physiology has been subject of investigation in many different species. High osmolarity is of importance for biotechnological production processes, where high cell densities and product titers are aspired. Several studies indicated that increased osmolarity of the growth medium can have a beneficial effect on recombinant protein production in different host organisms. Thus, the effect of osmolarity on the cellular physiology of Pichia pastoris, a prominent host for recombinant protein production, was studied in carbon limited chemostat cultures at different osmolarities. Transcriptome and proteome analyses were applied to assess differences upon growth at different osmolarities in both, a wild type strain and an antibody fragment expressing strain. While our main intention was to analyze the effect of different osmolarities on P. pastoris in general, this was complemented by studying it in context with recombinant protein production. RESULTS: In contrast to the model yeast Saccharomyces cerevisiae, the main osmolyte in P. pastoris was arabitol rather than glycerol, demonstrating differences in osmotic stress response as well as energy metabolism. 2D Fluorescence Difference Gel electrophoresis and microarray analysis were applied and demonstrated that processes such as protein folding, ribosome biogenesis and cell wall organization were affected by increased osmolarity. These data indicated that upon increased osmolarity less adaptations on both the transcript and protein level occurred in a P. pastoris strain, secreting the Fab fragment, compared with the wild type strain. No transcriptional activation of the high osmolarity glycerol (HOG) pathway was observed at steady state conditions. Furthermore, no change of the specific productivity of recombinant Fab was observed at increased osmolarity. CONCLUSION: These data point out that the physiological response to increased osmolarity is different to S. cerevisiae. Increased osmolarity resulted in an unfolded protein response (UPR) like response in P. pastoris and lead to pre-conditioning of the recombinant Fab producing strain of P. pastoris to growth at high osmolarity. The current data demonstrate a strong similarity of environmental stress response mechanisms and recombinant protein related stresses. Therefore, these results might be used in future strain and bioprocess engineering of this biotechnologically relevant yeast.

Project description:Basidiomycete high-redox potential laccases (HRPLs) working in human physiological fluids (pH 7.4, 150 mM NaCl) arise great interest in the engineering of 3D-nanobiodevices for biomedical uses. In two previous reports, we described the directed evolution of a HRPL from basidiomycete PM1 strain CECT 2971: i) to be expressed in an active, soluble and stable form in Saccharomyces cerevisiae, and ii) to be active in human blood. In spite of the fact that S. cerevisiae is suited for the directed evolution of HRPLs, the secretion levels obtained in this host are not high enough for further research and exploitation. Thus, the search for an alternative host to over-express the evolved laccases is mandatory.A blood-active laccase (ChU-B mutant) fused to the native/evolved ?-factor prepro-leader was cloned under the control of two different promoters (P(AOX1) and P(GAP)) and expressed in Pichia pastoris. The most active construct, which contained the P(AOX1) and the evolved prepro-leader, was fermented in a 42-L fed-batch bioreactor yielding production levels of 43 mg/L. The recombinant laccase was purified to homogeneity and thoroughly characterized. As happened in S. cerevisiae, the laccase produced by P. pastoris presented an extra N-terminal extension (ETEAEF) generated by an alternative processing of the ?-factor pro-leader at the Golgi compartment. The laccase mutant secreted by P. pastoris showed the same improved properties acquired after several cycles of directed evolution in S. cerevisiae for blood-tolerance: a characteristic pH-activity profile shifted to the neutral-basic range and a greatly increased resistance against inhibition by halides. Slight biochemical differences between both expression systems were found in glycosylation, thermostability and turnover numbers.The tandem-yeast system based on S. cerevisiae to perform directed evolution and P. pastoris to over-express the evolved laccases constitutes a promising approach for the in vitro evolution and production of these enzymes towards different biocatalytic and bioelectrochemical applications.

Dataset Information

Decreased expression of LRA4, a key gene involved in rhamnose metabolism, caused up-regulated expression of the genes in this pathway and autophagy in Pichia pastoris.

Publications

Decreased expression of LRA4, a key gene involved in rhamnose metabolism, caused up-regulated expression of the genes in this pathway and autophagy in Pichia pastoris.

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