Sequence-based factors influencing the expression of heterologous genes in the yeast Pichia pastoris-A comparative view on 79 human genes

Berlin University of Technology, Institute for Biotechnology, Department of Microbiology and Genetics, Gustav-Meyer-Allee 25, D-13355 Berlin, Germany.
Journal of Biotechnology (Impact Factor: 2.87). 06/2007; 130(1):1-10. DOI: 10.1016/j.jbiotec.2007.02.019
Source: PubMed


High yield expression of heterologous proteins is usually a matter of "trial and error". In the search of parameters with a major impact on expression, we have applied a comparative analysis to 79 different human cDNAs expressed in Pichia pastoris. The cDNAs were cloned in an expression vector for intracellular expression and recombinant protein expression was monitored in a standardized procedure and classified with respect to the expression level. Of all sequence-based parameters with a possible influence on the expression level, more than 10 were analysed. Three of those factors proved to have a statistically significant association with the expression level. Low abundance of AT-rich regions in the cDNA associates with a high expression level. A comparatively high isoelectric point of the recombinant protein associates with failure of expression and, finally, the occurrence of a protein homologue in yeast is associated with detectable protein expression. Interestingly, some often discussed factors like codon usage or GC content did not show a significant impact on protein yield. These results could provide a basis for a knowledge-oriented optimisation of gene sequences both to increase protein yields and to help target selection and the design of high-throughput expression approaches.

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Available from: Christine Lang, Oct 08, 2015
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    • "Until now, only a few applications of MBR using P. pastoris in bioprocess development can be found in literature, although several laboratories have been using microtiter plates for clone screening purposes [22,23]. "
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    ABSTRACT: In Pichia pastoris bioprocess engineering, classic approaches for clone selection and bioprocess optimization at small/micro scale using the promoter of the alcohol oxidase 1 gene (PAOX1), induced by methanol, present low reproducibility leading to high time and resource consumption. An automated microfermentation platform (RoboLector) was successfully tested to overcome the chronic problems of clone selection and optimization of fed-batch strategies. Different clones from Mut+ P. pastoris phenotype strains expressing heterologous Rhizopus oryzae lipase (ROL), including a subset also overexpressing the transcription factor HAC1, were tested to select the most promising clones.The RoboLector showed high performance for the selection and optimization of cultivation media with minimal cost and time. Syn6 medium was better than conventional YNB medium in terms of production of heterologous protein.The RoboLector microbioreactor was also tested for different fed-batch strategies with three clones producing different lipase levels. Two mixed substrates fed-batch strategies were evaluated. The first strategy was the enzymatic release of glucose from a soluble glucose polymer by a glucosidase, and methanol addition every 24 hours. The second strategy used glycerol as co-substrate jointly with methanol at two different feeding rates. The implementation of these simple fed-batch strategies increased the levels of lipolytic activity 80-fold compared to classical batch strategies used in clone selection. Thus, these strategies minimize the risk of errors in the clone selection and increase the detection level of the desired product.Finally, the performance of two fed-batch strategies was compared for lipase production between the RoboLector microbioreactor and 5 liter stirred tank bioreactor for three selected clones. In both scales, the same clone ranking was achieved. The RoboLector showed excellent performance in clone selection of P. pastoris Mut+ phenotype. The use of fed-batch strategies using mixed substrate feeds resulted in increased biomass and lipolytic activity. The automated processing of fed-batch strategies by the RoboLector considerably facilitates the operation of fermentation processes, while reducing error-prone clone selection by increasing product titers.The scale-up from microbioreactor to lab scale stirred tank bioreactor showed an excellent correlation, validating the use of microbioreactor as a powerful tool for evaluating fed-batch operational strategies.
    Microbial Cell Factories 03/2014; 13(1):36. DOI:10.1186/1475-2859-13-36 · 4.22 Impact Factor
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    • "However, heterologous protein secretion in S. cerevisiae is often not very efficient and limits the productivity to low levels. There are many factors affecting the protein titer including the protein properties (isoelectric point, distribution of AT-rich sequence in the cDNA, complex formation, post-translational modifications etc.) (Boettner et al. 2007), the leader sequences, the expression vector systems (promoters, selection markers, plasmid copy number etc.), and the folding and secretion capacity of the host cell. Many studies have focused previously on engineering the expression vector systems (Kjeldsen 2000; Liu et al. 2012; Seresht et al. 2012) and host strains to improve the protein yield and productivity (Idiris et al. 2010). "
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    ABSTRACT: The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high level over-expression of HSF1-R206S increased heterologous α-amylase yield 25 and 70 % when glucose was fully consumed, and 37 and 62 % at the end of the ethanol phase, respectively. Moderate and high level over-expression also improved endogenous invertase yield 118 and 94 %, respectively. However, human insulin precursor was only improved slightly and this only by high level over-expression of HSF1-R206S, supporting our previous findings that the production of this protein in S. cerevisiae is not limited by secretion. Our results provide an effective strategy to improve protein secretion and demonstrated an approach that can induce ER and cytosolic chaperones simultaneously.
    Applied Microbiology and Biotechnology 12/2012; 97(8). DOI:10.1007/s00253-012-4596-9 · 3.34 Impact Factor
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    • "A lack of correlation between successful protein production and gene/protein sequence characteristics indicates that there are as yet unknown sequence features that complicate the heterologous expression of plant genes in P. pastoris. The 50% success rate observed with barley XTH genes seems to reflect that observed for human genes (44 of 79 showing no expression) (Boettner et al. 2007), and supports anecdotal evidence from our own laboratory obtained with the heterologous expression of dicot XTH genes in P. pastoris. Clearly, further in-depth analysis correlating sequence or structural features with failed heterologous expression in the P. pastoris system will be required to improve the success rate through sequence engineering. "
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    ABSTRACT: Heterologous expression of plant genes, particularly those encoding carbohydrate-active enzymes such as glycoside hydrolases and glycosyl transferases, continues to be a major hurdle in the functional analysis of plant proteomes. Presently, there are few convenient systems for the production of recombinant plant enzymes in active form and at adequate levels for biochemical and structural characterization. The methylotrophic yeast Pichia pastoris is an attractive expression host due to its ease of manipulation and its capacity to perform post-translational protein modifications, such as N-glycosylation [Daly and Hearn (2005) J Mol Recognit 18: 119-138]. Here, we demonstrate the utility of the P. pastoris SMD1168H/pPICZ-alpha C system for the expression of a range of xyloglucan endo-transglycosylase/hydrolase (XTH) cDNAs from barley (Hordeum vulgare). Although stable transformants were readily obtained by positive selection for vector-induced antibiotic resistance for all of the nine constructs tested, only five isoforms were secreted as soluble proteins into the culture medium, four in active form. Furthermore, production levels of these five isoforms were found to be variable, depending on the transformant, which further underscores the necessity of screening multiple clones for expression of active enzyme. Failure to express certain XTH isoforms in P. pastoris could not be correlated with any conserved gene or protein sequence properties, and this precluded using rational sequence engineering to enhance heterologous expression of the cDNAs. Thus, while significant advances are reported here, systems for the heterologous production of plant proteins require further development.
    Plant Biotechnology Journal 01/2010; 27(JSPS Sweden Colloquium-No. 3):251-258. DOI:10.5511/plantbiotechnology.27.251 · 5.75 Impact Factor
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