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Xylanase, an enzyme capable of hydrolyzing non-starch polysaccharides found in grain structures like wheat, has been found to improve the organizational structure of dough and thus increase its volume. In our past work, one promising xylanase FXYL derived from Fusarium oxysporum Fo47 and first expressed 779.64 U/mL activity in P. pastoris. It has shown significant potential in improving the quality of whole wheat bread, making it become a candidate for development as a new flour improver. After optimization of expression elements and gene dose, the xylanase activity of FXYL strain carrying three-copies reached 4240.92 U/mL in P. pastoris. In addition, 12 factors associated with the three stages of protein expression pathway were co-expressed individually in order in three-copies strain, and the translation factor Pab1 co-expression increased FXYL activity to 8893.53 U/mL. Nevertheless, combining the most effective or synergistic factors from three stages did not exhibit better results than co-expressing them alone. To further evaluate the industrial potential, the xylanase activity and protein concentration reached 81184.51 U/mL and 11.8 g/L in a 5 L fed-batch fermenter. These engineering strategies improved the expression of xylanase FXYL by more than 104-fold, providing valuable insights for the cost-effective industrial application of FXYL in the baking field.

The production of heterologous proteins via recombinant expression system is a focus of genetic engineering research. Because of its ease of genetic manipulation, high degree of heterologous protein secretion, and post-translational modification, Pichia pastoris, a methylotrophic yeast, has become an essential strain for protein production in industrial applications and is frequently employed in the manufacturing of enzymes. Although some heterologous proteins can be expressed in P. pastoris, the amount of expression is modest and has to be increased. Therefore, it is critical to investigate the principle and method of boosting the expression of heterologous proteins in P. Pastoris in order to lower industrial production costs and improve economic benefits. In this review, the optimizing strategies for improving heterologous protein expression in P. Pastoris in recent years are summarized from six perspectives: Gene level, transcription level, translation level, protein folding and secretion levels, cell resistance level, and fermentation technology, with the goal of demonstrating comprehensively how to improve the expression of heterologous protein in P. Pastoris expression system. [ABSTRACT FROM AUTHOR]

The production of heterologous proteins via microbial secretion is currently a prominent focus in biological product development. However, the full realization of this method remains hindered by limited understanding of the protein secretion mechanism with in target microorganisms. To enhance protein production, microbial chassis cell are being reinforced via synthetic biology and metabolic engineering, proving highly efficient. Among microorganisms, gram-positive bacteria are widely employed due to their absence of an outer membrane, facilitating the production of various secreted proteins, including industrial enzymes, functional nutrients, and medical peptides. Fungi also play a pivotal role in secreted protein production, thanks to their efficient post-translational modification and secretory capabilities. In this context, we highlighted the secretion mechanism of heterologous proteins in B. subtilis and P. pastoris, two classical secretory systems. Their proficient protein secretion, low number of extracellular heteroproteins, and convenient genetic manipulation make them both highly preferable as chassis cells. On this basis, the strategies of enhancing protein secretory pathway after synthesis through genetic engineering were elaborated, and the application of some cutting-edge technologies in this pathway were expanded.

Abstract Background Translational regulation played an important role in the correct folding of heterologous proteins to form bioactive conformations during biogenesis. Translational pausing coordinates protein translation and co-translational folding. Decelerating translation elongation speed has been shown to improve the soluble protein yield when expressing heterologous proteins in industrial expression hosts. However, rational redesign of translational pausing via synonymous mutations may not be feasible in many cases. Our goal was to develop a general and convenient strategy to improve heterologous protein synthesis in Pichia pastoris without mutating the expressed genes. Results Here, a large-scale deletion library of ribosomal protein (RP) genes was constructed for heterologous protein expression in Pichia pastoris, and 59% (16/27) RP deletants have significantly increased heterologous protein yield. This is due to the delay of 60S subunit assembly by deleting non-essential ribosomal protein genes or 60S subunit processing factors, thus globally decreased the translation elongation speed and improved the co-translational folding, without perturbing the relative transcription level and translation initiation. Conclusion Global decrease in the translation elongation speed by RP deletion enhanced co-translational folding efficiency of nascent chains and decreased protein aggregates to improve heterologous protein yield. A potential expression platform for efficient pharmaceutical proteins and industrial enzymes production was provided without synonymous mutation.

Objective: Translational regulation plays an important role in protein synthesis. Our goal was to screen translation-related factors to improve heterologous protein expression in Pichia pastoris. Results: Twenty-eight translation-related factors were overexpressed in P. pastoris GS115 expressing enhanced green fluorescent protein (eGFP). The results showed that overexpression of Bcy1, the regulatory subunit of protein kinase A (PKA), significantly increased both eGFP expression and cell biomass by 20% under methanol induction for 120 h. Additionally, overexpression of Bcy1 elevated the growth rate by 18% and increased production of the industrial enzyme Phytase (Phy) by 26%. Transcriptome analysis indicated that the overall expression of ribosomal protein genes was significantly downregulated and that postdiauxic shift genes and stress response element genes were upregulated. Conclusions: Bcy1 regulates ribosome protein genes, postdiauxic shift genes and stress response element genes, leading to improved cell growth and heterologous protein expression. This study provides a convenient and universal factor for heterologous protein production. [ABSTRACT FROM AUTHOR]

• Novel P. pastoris endogenous signal peptides and folding factors were predicted based on the reported secretome and genome and tested experimentally. • The signal sequence Msb2 increased the secretory production of all 3 reporter proteins. • The signal sequence Dan4 enhanced total protein production of all reporter proteins up to 172-fold. • New folding factors (Mpd1p, Pdi2p, and Sil1p) exhibited a protein-specific effect on cell growth, transcription and expression of different reporter proteins. • These novel signal peptides and folding factors can be used for promoting secretion of heterologous proteins in P. pastoris. Secretory expression is most often desired but usually hampered by limitations of signal peptide processing and protein folding in the methylotrophic yeast Pichia pastoris. To alleviate such limitations, novel endogenous signal peptides (Dan4, Gas1, Msb2, and Fre2) and folding factors (Mpd1p, Pdi2p, and Sil1p) were predicted based on the reported P. pastoris secretome and genome. Their effects were investigated using three reporter proteins: yeast-enhanced green fluorescent protein (yEGFP), β-galactosidase (Gal) and cephalosporin C acylase (SECA), in comparison with the commonly used Saccharomyces cerevisiae alpha-mating factor pre-pro leader sequence (α-MF) or folding factors (Pdi1p, BiP, and Hac1p). The newly identified signal sequences were superior over α-MF for production of heterologous proteins. The signal peptide Msb2 increased the specific extracellular production of all reporter proteins, ranging from 1.5- to 8.0-fold, and Dan4 enhanced all total protein production up to 172-fold. Co-expression of folding factors exhibited a protein-specific effect on cell growth, transcription and expression of different reporter genes. All of the novel folding factors enhanced total production of SECA, and Sil1p performed best in the extracellular SECA production, showing a 3.3-fold increase. These novel signal peptides and folding factors can be used for promoting secretion of heterologous proteins in P. pastoris. [ABSTRACT FROM AUTHOR]

The yeast Pichia pastoris has been widely used as a host system for heterologous protein production, such as; antibody fragments, recombinant huridina, recombinant human insulin and recombinant human erythropoietin. Its wide application is due to the availability of the promoter of alcohol oxidase enzyme, which is strong and regulatable by methanol and its ability to reach high cell densities in bioreactors cheap culture media. This review aims to provide information about yeast P. pastoris as suitable host system for heterologous protein production. The majority of the P. pastoris strains that are employed in the production of heterologous proteins possess as auxotrophic selection marker the histidinol dehydrogenase gene (his4), which is essential for synthesis of histidine amino acid. As this yeast is capable of using methanol as carbon source and energy, most of its expression vectors are based on promoters of enzymes involved in the methanol metabolism among which is the alcohol oxidase 1 (AOX1). The strain selection, vectors and optimization of culture conditions for a bioprocess with P. pastoris is essential in the obtaining of high levels of the heterologous protein. [ABSTRACT FROM AUTHOR]

Biofiltration is a technology for the biological degradation of multiple gas pollutants. It consists of a biological active bed where the contaminant gas is vented through and aerobically degraded. In this study, a novel methanol biofiltration process coupled with the production of the heterologous protein Endochitinase-42, using a genetically modified Pichia pastoris strain, was investigated. Three identical biofilters of 3.3 L packed with Perlite were operated under discontinuing addition of nutritive solution. Four important parameters such as methanol inlet loading rate (ILR), pH, nitrogen concentration, and empty bed residence time (EBRT) were studied. Evaluation of ILR resulted in a maximum elimination capacity (EC) of 643 g m −3 h −1 and heterologous Ech42 activity of 1020 U L −1 . In another stage, it was observed that nitrogen is a limiting factor not only for methanol degradation, but also for protein production, being 7.5 g L −1 ammonium sulfate (7 gC/gN) the optimal value with a protein activity of 1172 U L −1 . Moreover, pH 5.5 and EBRT of 60 s were the most favorable values for enzymatic production. In general, a set of operational conditions, including 7.5 g L −1 ammonium sulfate, pH 5, and EBRT of 60 s were optimal to maximize the enzymatic productivity. [ABSTRACT FROM AUTHOR]

The available promoters in the Pichia pastoris expression platform are still limited. We selected and identified a novel strong constitutive promoter, P, and tested its promoter activity using enhanced green fluorescent protein (EGFP) as a reporter. Potential promoter regions of P were cloned upstream of the EGFP gene and promoter activity was analyzed by measuring fluorescence intensity. P exhibited significantly stronger promoter activity than the classic strong constitutive promoters P and P under various carbon sources, suggesting that P is a strong and constitutive promoter. Hence, P can be used as a promoter for high-level expression of heterologous proteins. [ABSTRACT FROM AUTHOR]

Protein translation, translocation, folding, processing, and secretion in eukaryotic cells are complex and not always straightforward processes, e.g., different routes of secretion and degradation exist. Formation of malfolded proteins in the endoplasmic reticulum (ER) can be one of the major bottlenecks for recombinant protein production. In this regard, an in-depth analysis of the interactions of a secreted protein during its pathway through the cell may be beneficial, as realized in this study for the methylotrophic yeast Pichia pastoris. The antibody fragment Fab3H6 used here is the anti-idiotype to the HIV neutralizing antibody 2F5 and is known to be intracellularly degraded in significant amounts when expressed in P. pastoris. The interactome of Fab3H6 was analyzed by using a pull-down mass spectrometry approach, and 23 proteins were found to bind specifically to the antibody fragment. Those allowed concluding that Fab3H6 is post-translationally translocated into the ER and degraded via the proteasome as well as the vacuole. In line with this, the expression of Fab3H6 increased the proteasomal activities by over 20%. Partial inhibition of the proteasome resulted in a significant increase of extracellular Fab3H6. Thus, it seems that ER quality control overshoots its requirements for the recombinant protein expressed and that more than just terminally malfolded protein is degraded by ER-associated degradation. This work will further facilitate our understanding how recombinant proteins behave in the secretory pathway. [ABSTRACT FROM AUTHOR]

Systems biotechnology has been established as a highly potent tool for bioprocess development in recent years. The applicability to complex metabolic processes such as protein synthesis and secretion, however, is still in its infancy. While yeasts are frequently applied for heterologous protein production, more progress in this field has been achieved for bacterial and mammalian cell culture systems than for yeasts. A critical comparison between different protein production systems, as provided in this review, can aid in assessing the potentials and pitfalls of applying systems biotechnology concepts to heterologous protein producing yeasts. Apart from modelling, the methodological basis of systems biology strongly relies on postgenomic methods. However, this methodology is rapidly moving so that more global data with much higher sensitivity will be achieved in near future. The development of next generation sequencing technology enables an unexpected revival of genomic approaches, providing new potential for evolutionary engineering and inverse metabolic engineering. [ABSTRACT FROM AUTHOR]

The Pichia pastoris expression system has been successfully used for production of various recombinant heterogeneous proteins. The productivity of P. pastoris can be improved substantially by bioreactor cultivations. However, heterologous proteins degradation increases as well in high-cell density culture. Proteolytic degradation is a serious problem since the yeast has been employed to express recombinant proteins. In this review, some of the recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels on the cytosolic proteasome, vacuolar proteases, and proteases located within the secretory pathway in P. pastoris, are reviewed. [ABSTRACT FROM AUTHOR]

This paper describes the establishment of flow cytometric methods for recombinant Pichia pastoris strains, and their application to a lab scale fed batch fermentation. Using a strain which secretes human trypsinogen, the viability and the product which remained associated to the cell were measured with propidium iodide and immunofluorescent staining, respectively. Viability decreases significantly below 70% during the methanol fed batch phase, indicating a stress situation triggered by the fermentation conditions. Cell associated product is accumulated earlier after methanol induction than secreted product. These data demonstrate that flow cytometry is a powerful tool for the analysis and optimization of recombinant protein production processes, and they indicate the need to further improve a widely used fermentation protocol for P. pastoris. [Copyright &y& Elsevier]

Background: Translational regulation played an important role in the correct folding of heterologous proteins to form bioactive conformations during biogenesis. Translational pausing coordinates protein translation and co-translational folding. Decelerating translation elongation speed has been shown to improve the soluble protein yield when expressing heterologous proteins in industrial expression hosts. However, rational redesign of translational pausing via synonymous mutations may not be feasible in many cases. Our goal was to develop a general and convenient strategy to improve heterologous protein synthesis in Pichia pastoris without mutating the expressed genes. Results: Here, a large-scale deletion library of ribosomal protein (RP) genes was constructed for heterologous protein expression in Pichia pastoris, and 59% (16/27) RP deletants have significantly increased heterologous protein yield. This is due to the delay of 60S subunit assembly by deleting non-essential ribosomal protein genes or 60S subunit processing factors, thus globally decreased the translation elongation speed and improved the co-translational folding, without perturbing the relative transcription level and translation initiation. Conclusion: Global decrease in the translation elongation speed by RP deletion enhanced co-translational folding efficiency of nascent chains and decreased protein aggregates to improve heterologous protein yield. A potential expression platform for efficient pharmaceutical proteins and industrial enzymes production was provided without synonymous mutation. [ABSTRACT FROM AUTHOR]