Protein Production In Pichia Pastoris Research Articles

Expression and Purification of Cp3GT: Structural Analysis and Modeling of a Key Plant Flavonol-3-O Glucosyltransferase from Citrus paradisi.

Glycosyltransferases (GTs) are pivotal enzymes in the biosynthesis of various biological molecules. This study focuses on the scale-up, expression, and purification of a plant flavonol-specific 3-O glucosyltransferase (Cp3GT), a key enzyme from Citrus paradisi, for structural analysis and modeling. The challenges associated with recombinant protein production in Pichia pastoris, such as proteolytic degradation, were addressed through the optimization of culture conditions and purification processes. The purification strategy employed affinity, anion exchange, and size exclusion chromatography, leading to greater than 95% homogeneity for Cp3GT. In silico modeling, using D-I-TASSER and COFACTOR integrated with the AlphaFold2 pipeline, provided insights into the structural dynamics of Cp3GT and its ligand binding sites, offering predictions for enzyme-substrate interactions. These models were compared to experimentally derived structures, enhancing understanding of the enzyme's functional mechanisms. The findings present a comprehensive approach to produce a highly purified Cp3GT which is suitable for crystallographic studies and to shed light on the structural basis of flavonol specificity in plant GTs. The significant implications of these results for synthetic biology and enzyme engineering in pharmaceutical applications are also considered.

Alternative secretory signal sequences for recombinant protein production in Pichia pastoris

Extracellular protein production is primarily preferred to facilitate the downstream processes in recombinant protein production. Secretion of recombinant proteins is mediated by the processing of signal peptides in their N-terminal portion by the secretory mechanism of host expression systems. These molecular elements involved in secretion are functionally interchangeable between different species and secretion sequence screening is one of the widely used approaches to improve extracellular protein production. In this study, α-mating and protein internal repeats (PIR) secretion sequences isolated from different yeasts (Kluyveromyces lactis, Kluyveromyces marxianus and Hansenula polymorpha) were tested in Pichia pastoris for the production of two different proteins (α-amylase and xylanase) and compared with the well-known secretory signals, S. cerevisiae α-mating factor (Sc-MF) and P. pastoris protein internal repeats PIR (PpPIR). The results obtained showed the potential of signal sequences tested. Among the tested peptides, the highest yields were achieved with H. polymorpha protein internal repeats (HpPIR) and K. lactis α-mating factor (Kl-MF) for xylanase and K. marxianus protein internal repeats (KmPIR) and K. lactis α-mating factor (Kl-MF) for amylase. In further studies, these sequences can be evaluated as alternatives in the production of different proteins in P. pastoris and in the production of recombinant proteins in different expression systems.

Applicability of the heterologous yeast promoters for recombinant protein production in Pichia pastoris.

Promoter choice is an important step in recombinant protein production, which directly determines the expression manner as constitutive or inducible and the expression level of the recombinant protein. This study aims to investigate the applicability of heterologous yeast promoters (Kluyveromyces marxianus TPI, Hansenula polymorpha PMA, Candida tropicalis ICL, and Saccharomyces cerevisiae CUP) in Pichia pastoris. The regulation mode of the CtICL and ScCUP promoters in P. pastoris was found to be inducible and that of the KmTPI and HpPMA was constitutive. The carbon sources in which the promoters exhibited the highest activity were determined as glycerol for PMA and TPI, glucose for CUP, and ethanol for ICL. The DNA region showing the highest activity was determined as 1000bp for all promoters by promoter deletion analysis. Results from the study demonstrate the potential of inducible and constitutive heterologous promoters allowing expression under different conditions in the P. pastoris expression system and offers alternatives to frequently used promoters. KEY POINTS: • Heterologous promoters exhibited similar expression pattern in P. pastoris with its native host. • HpPMA has the highest promoter activity among the heterologous promoters tested. • Reporter gene expression with ScCUP is responsive to elevating Cu2+in P. pastoris.

Oxidative stress tolerance contributes to heterologous protein production in Pichia pastoris

BackgroundPichia pastoris (syn. Komagataella phaffii) is an important yeast system for heterologous protein expression. A robust P. pastoris mutant with oxidative and thermal stress cross-tolerance was acquired in our previous study. The robust mutant can express a 2.5-fold higher level of lipase than its wild type (WT) under methanol induction conditions.ResultsIn this study, we found that the robust mutant not only can express a high level of lipase, but also can express a high level of other heterogeneous proteins (e.g., green fluorescence protein) under methanol induction conditions. Additionally, the intracellular reactive oxygen species (ROS) levels in the robust mutant were lower than that in the WT under methanol induction conditions. To figure out the difference of cellular response to methanol between the WT and the robust mutant, RNA-seq was detected and compared. The results of RNA-seq showed that the expression levels of genes related to antioxidant, MAPK pathway, ergosterol synthesis pathway, transcription factors, and the peroxisome pathway were upregulated in the robust mutant compared to the WT. The upregulation of these key pathways can improve the oxidative stress tolerance of strains and efficiently eliminate cellular ROS. Hence, we inferred that the high heterologous protein expression efficiency in the robust mutant may be due to its enhanced oxidative stress tolerance. Promisingly, we have indeed increased the expression level of lipase up to 1.6-fold by overexpressing antioxidant genes in P. pastoris.ConclusionsThis study demonstrated the impact of methanol on the expression levels of genes in P. pastoris and emphasized the contribution of oxidative stress tolerance on heterologous protein expression in P. pastoris. Our results shed light on the understanding of protein expression mechanism in P. pastoris and provided an idea for the rational construction of robust yeast with high expression ability.

Ethanol fed-batch bioreactor operation to enhance therapeutic protein production in Pichia pastoris under hybrid-architectured ADH2 promoter

ABSTRACT We presented ethanol fed-batch bioreactor operations (FBBOs) designed for enhanced recombinant human growth hormone (rhGH) production in Pichia pastoris constructed with novel hybrid-architectured ADH2 promoter, PADH2-Cat8-L2. The parameters in the fed-batch fermentation of ethanol were investigated, and the boundaries of the production domain in terms of the design parameters were determined. Two FBBO design methods were used, and a platform aiming to adjust cellular metabolism for the generation of constant specific growth rate (μ) and substrate concentration (CEtOH) was established. The highest overall productivity (rP,ov) was generated with model-based continuous feed-stream (CFS) design with μpreD = 0.035 h-1 up to tmax ≤ 9 h as rP,ov = 3.0 mg L-1 h-1 and CrhGH = 34 mg L-1. The ethanol-stat FBBO at CEtOH,set = 0.5 g L-1 allowed increased product titer with high productivity and the highest CrhGH =90.1 mg L-1 at tmax = 24 h. The effect of CEtOH on specific rates for cell reactions was represented by the Haldane model in which the cell generation and rhGH production were simulated with low mean relative errors of 6.5 and 16%, respectively. The results indicate the need for an upgraded CFS design model with kinetic expressions involving substrate inhibition. We conclude that a two-step hybrid fed-batch design strategy to be started (i) with the initial-period of the model-based FBBO, in turn, proceeded (ii) with the controlled feeding with ethanol-stat FBBO enables a gradual approach to the hypothetical performance of FBBOs.

Engineering the regulatory site of the catalase promoter for improved heterologous protein production in Pichia pastoris.

To build a stronger Pichia pastoris PCAT1 promoter and to identify putative transcriptional factor binding sites (TFBSs) on PCAT1 that affect the activity of the promoter. A synthetic library of PCAT1 was generated by deleting or duplicating putative TFBS motifs in the promoter sequence. CSRE, MIG1, RAP1 and HAP2/3/4 were found to have important effects on PCAT1 activity. The PCAT1 variant P4 with a putative binding site of RAP1 on the promoter sequence showed a stronger activity compared with that of the wild-type PCAT1 and PAOX1, which is the strongest natural P. pastoris promoter that has been reported. This inference was confirmed with EGFP (enhanced green fluorescent protein) and Candida Antarctica lipase B as the reporters. The role of the transcriptional regulator RAP1 may be important in PCAT1 methanol induction. A stronger PCAT1 variant can be constructed by the duplication of the putative binding site of RAP1 on the PCAT1 promoter sequence. This PCAT1 variant has potential value for heterologous protein production, metabolic engineering, and synthetic biology.

Redox Engineering by Ectopic Overexpression of NADH Kinase in Recombinant Pichia pastoris (Komagataella phaffii): Impact on Cell Physiology and Recombinant Production of Secreted Proteins.

High-level expression and secretion of heterologous proteins in yeast cause an increased energy demand, which may result in altered metabolic flux distributions. Moreover, recombinant protein overproduction often results in endoplasmic reticulum (ER) stress and oxidative stress, causing deviations from the optimal NAD(P)H regeneration balance. In this context, overexpression of genes encoding enzymes catalyzing endogenous NADPH-producing reactions, such as the oxidative branch of the pentose phosphate pathway, has been previously shown to improve protein production in Pichia pastoris (syn. Komagataella spp.). In this study, we evaluate the overexpression of the Saccharomyces cerevisiaePOS5-encoded NADH kinase in a recombinant P. pastoris strain as an alternative approach to overcome such redox constraints. Specifically, POS5 was cooverexpressed in a strain secreting an antibody fragment, either by directing Pos5 to the cytosol or to the mitochondria. The physiology of the resulting strains was evaluated in continuous cultivations with glycerol or glucose as the sole carbon source, as well as under hypoxia (on glucose). Cytosolic targeting of Pos5 NADH kinase resulted in lower biomass-substrate yields but allowed for a 2-fold increase in product specific productivity. In contrast, Pos5 NADH kinase targeting to the mitochondria did not affect growth physiology and recombinant protein production significantly. Growth physiological parameters were in silico evaluated using the recent upgraded version (v3.0) of the P. pastoris consensus genome-scale metabolic model iMT1026, providing insights on the impact of POS5 overexpression on metabolic flux distributions.IMPORTANCE Recombinant protein overproduction often results in oxidative stress, causing deviations from the optimal redox cofactor regeneration balance. This becomes one of the limiting factors in obtaining high levels of heterologous protein production. Overexpression of redox-affecting enzymes has been explored in other organisms, such as Saccharomyces cerevisiae, as a means to fine tune the cofactor regeneration balance in order to obtain higher protein titers. In the present work, this strategy is explored in P. pastoris In particular, one NADH kinase enzyme from S. cerevisiae (Pos5) is used, either in the cytosol or in mitochondria of P. pastoris, and its impact on the production of a model protein (antibody fragment) is evaluated. A significant improvement in the production of the model protein is observed when the kinase is directed to the cytosol. These results are significant in the field of heterologous protein production in general and in particular in the development of improved metabolic engineering strategies for P. pastoris.

Continuous operation, a realistic alternative to fed-batch fermentation for the production of recombinant lipase B from Candida antarctica under the constitutive promoter PGK in Pichia pastoris

In this work, continuous and fed-batch operational modes for producing the recombinant protein lipase B from Candida antarctica under the new constitutive promoter PGK in the cell factory Pichia pastoris was assessed. For this purpose, the influence of the specific growth rate (μ) was examined from 0.05 to 0.16 h−1 on various key bioprocess parameters.The overall biomass-to-substrate yield YX/S and specific substrate uptake rate (qs) were found to be independent of the particular operational mode. However, the μ-dependence of specific production rate (qp) differed with the cultivation strategy used. Thus, qp was linearly related in the continuous mode but suggested saturation in fed-batch cultures.The highest volumetric and specific productivity were obtained at the highest μlevels in the continuous mode, which provided volumetric and specific productivities (QP and Qe, respectively) roughly 1.5 and 3 times greater than the fed-batch mode. The continuous mode also proved more effective in the long rung; thus, CalB production after 6 weeks was estimated to be about 5.8 times greater than with the fed-batch mode.Continuous operation with the novel constitutive promoter PGK is thus a realistic alternative to the standard fed-batch cultivation at a high cell density typically used in industrial bioprocesses for heterologous protein production in Pichia pastoris.

Analyzing Recombinant Protein Production in Pichia pastoris with Targeted Proteomics.

New mass spectrometry approaches enable antibody-independent tracking of protein production. Herein, we outline an antibody-independent mass spectrometry method for tracking recombinant protein production in the methylotrophic yeast Pichia pastoris system.

The growth of Pichia pastoris Mut+ on methanol-glycerol mixtures fits to interactive dual-limited kinetics: model development and application to optimised fed-batch operation for heterologous protein production.

The methanol-glycerol co-feeding during the induction stage for heterologous protein production in Pichia pastoris has shown significant productive applications. Available model analysis applied to this dual-limited condition is scarce and normally does not consider the interaction effects between the substrates. In this work, a dual-limited growth model of P. pastoris considering an interactive kinetic effect was applied to an optimised fed-batch process production of heterologous Rhizopus oryzae lipase (ROL). In the proposed model, the growth kinetics on glycerol is fully expressed, whereas methanol kinetics is modulated by the co-metabolisation of glycerol, resulting in an enhancing effect of glycerol-specific growth rate. The modelling approach of fed-batch cultures also included the methanol volatilisation caused by the aeration that was found to be a not-negligible phenomenon. The model predicts the ability of P. pastoris to keep control of the methanol concentration in the broth during ROL-optimised production process in fed batch and fits satisfactorily the specific cell growth rate and ROL production. Implications of interaction effect are discussed applying the general procedure of modelling approach.

Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose-regulated GTH1 promoter of Pichia pastoris.

Protein production in Pichia pastoris is often based on the methanol‐inducible P AOX1 promoter which drives the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties such as the glucose‐regulated P GTH1 promoter which we reported recently. To further increase its potential, we investigated its regulation in more details by the screening of promoter variants harboring deletions and mutations. Thereby we could identify the main regulatory region and important putative transcription factor binding sites of P GTH1. Concluding from that, yeast metabolic regulators, monomeric Gal4‐class motifs, carbon source‐responsive elements, and yeast GC‐box proteins likely contribute to the regulation of the promoter. We engineered a P GTH1 variant with greatly enhanced induction properties compared with that of the wild‐type promoter. Based on that, a model‐based bioprocess design for high volumetric productivity in a limited time was developed for the P GTH1 variant, to employ a glucose fed‐batch strategy that clearly outperformed a classical methanol fed‐batch of a P AOX1 strain in terms of titer and process performance.

Naturally occurring novel promoters around pyruvate branch-point for recombinant protein production in Pichia pastoris (Komagataella phaffii): Pyruvate decarboxylase- and pyruvate kinase- promoters

Novel strong promoter discovery is crucial for the design of novel strains of the industrial yeast Pichia pastoris for recombinant protein (r-protein) production. In order to remedy the deficiency, transcriptome and proteome data of P. pastoris were analysed. Genes having higher expression levels than glyceraldehyde-3-phosphate-dehydrogenase (GAP) gene were identified as promoter sources. Pyruvate kinase- (PPYK) and pyruvate decarboxylase- (PPDC) promoters around pyruvate-node were determined as promising candidates, and in silico analysis of the putative promoter regions was performed. The putative promoters were introduced into the plasmid pGAPZαA::hGH harboring human growth hormone (hGH) gene, instead of PGAP. Single-copy hGH gene carrying strains under PPYK, PPDC, and for comparison under PGAP were tested in high-cell-density rhGH fermentations, respectively, abbreviated as BRPYK, BRPDC, and BRGAP. Comparative promoter strength assessment was made by the mRNA-transcription-copy-number (mTCN) and r-protein concentration measurements, and by constrained flux analysis. PPDC and PPYK exhibited higher activity compared to PGAP. Highest rhGH titer was obtained in BRPDC as 122 mg dm−3 at t = 15 h, in BRPYK as 101 mg dm−3 at t = 12 h, and in BRGAP as 58 mg dm−3 at t = 9 h; while, with PPDC and PGAP the mTCNs were high and close. The flux distributions demonstrated regulatory effects of the novel promoters in the engineered strains and validated cross-pathway regulatory interactions.

Presence of protein production enhancers results in significantly higher methanol-induced protein production in Pichia pastoris

BackgroundThe yeast Komagataella phaffii, better known as Pichia pastoris, is a commonly used host for recombinant protein production. Here expression vectors are reported that address the different steps of the transcription–translation–secretion pathway of heterologous protein production.ResultsTranscription and translation enhancing elements were introduced in an expression cassette for the production of recombinant Aspergillus niger feruloyl esterase A. The yield was increased by threefold as compared to the yield without these elements. Multiple copy strains were selected using a zeocin resistance marker in the expression cassette and showed another sixfold higher yield. Modification of the C-terminal amino acid sequence of the secretion signal did not significantly improve the production yield. Similar data were obtained for the production of another protein, recombinant human interleukin 8. Upscaling to fed-batch fermentation conditions resulted in a twofold increase for reference strains, while for strains with enhancing elements a tenfold improvement was observed.ConclusionsPichia pastoris is used for recombinant protein production in industrial fermentations. By addressing the transcription and translation of mRNA coding for recombinant protein, significant yield improvement was obtained. The yield improvement obtained under microscale conditions was maintained under fed-batch fermentation conditions. These data demonstrate the potential of these expression vectors for large scale application as improved production of proteins has major implications on the economics and sustainability of biocatalyst dependent production processes e.g. for the production of pharmaceuticals and for the bioconversions of complex molecules.

Strategies for cloning and expression of a codon‐optimized human P‐glycoprotein (MDR1) in the yeast Pichia pastoris

P‐glycoprotein (P‐gp) is an ABC transporter which actively transports xenobiotics out of cells. Overexpression of P‐gp is one of the main causes for the development of multidrug resistances (MDR) in cancer and other diseases. Because of its ability to bind and transport a large variety of chemically different compounds, overexpression of P‐gp in these MDR cancers leads to resistances against chemotherapeutic drugs that are distinctly different in structure, chemical composition, and functionality. Using high‐throughput in silico ligand docking studies, we have previously identified drug‐like compounds that inhibit P‐gp and cause reversal of MDR in a prostate cancer cell line (Brewer et al., Mol. Pharmacol. 86, 716–726, 2014; Follit et al., Pharmacol. Res. Perspect. 3, e00170, 2015). To analyze the mechanism of inhibition by these initially identified P‐gp inhibitors as well as newly discovered drug‐like compounds that reversed MDR in cancers, biochemical and biophysical analysis of inhibitor action is imperative. For this reason, a gene for human P‐gp (MDR1) was designed with optimized codon usage for translation in P. pastoris. A pPinkHC‐MDR1 plasmid was created for integration into Pichia pastoris PichiaPink™, a commercially available high yield expression system that includes specific strains with protease knock outs to limit protein degradation. Successful MDR1 DNA integration in the PichiaPink™ chromosome was confirmed, but significant protein expression has thus far not been observed. We have re‐cloned the codon optimized MDR1 DNA into another Pichia expression vector, pHIL. This vector allows integration of the gene of interest into the Pichia pastoris strain, GS115, which has been used successfully by us in the past to express a mouse cysteine‐less P‐glycoprotein. The resulting colonies displayed a Muts phenotype, which is indicative of the correct recombination into the Pichia genomic DNA. Initial Western blot analyses using both P‐gp specific and His‐tag specific antibodies showed that after 24 hours of growth in minimal methanol media the MDR1 GS115 clone did express reasonable amounts of human P‐gp. We report here our attempts at constructing a stable, codon optimized, human P‐gp gene for protein production in Pichia pastoris as well as attempts to express, purify and characterize the human P‐glycoprotein.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, Ms. Lynn McBee and New England Biolabs, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation of Sorbitol-Methanol Co-Feeding Strategy on Production of Recombinant Human Growth Hormone in Pichia Pastoris.

Recombinant protein production in Pichia pastoris is based on alcohol oxidase promoters which are regulated by methanol. However, the use of methanol has several disadvantages, which is why current trends in bioprocess development with Pichia pastoris (P. pastoris) are focusing on methanol mixed feeding strategies. This work aimed to develop a new experimental method and compare the effect of various fractions of sorbitol in mixed feeding strategy with stepwise addition of methanol to maximize the productivity of human growth hormone. Accordingly, fed-batch culture performed with a mixed feed of sorbitol/methanol. Sorbitol at concentrations of 30, 40, 50 and 60 gram per liter was added in batch-wise mode to the medium at the beginning of induction phase and the rate of methanol addition was increased stepwise during the first 12 h of production and then kept constant. In order to understand the effects of various co-substrate feeding strategies on P. pastoris and its production of hGH, cell mass, dry cell weight, total protein, hGH expression level and hGH concentration were analyzed and the results compared with the basic feeding protocol using one-way analysis of variance (ANOVA). According to the obtained results, sorbitol at a concentration of 50 g/L could significantly increase the production yield. Under such optimal conditions cell biomass was 108 g/L (DCW), total protein was 0.807 g/L, expression level was 83.1% and rhGH concentration was 0.667 g/L following 30 h induction.

Improvement of extracellular bacterial protein production in Pichia pastoris by co-expression of endoplasmic reticulum residing GroEL–GroES

Pichia pastoris is an established host system for heterologous protein expression. However, the potential productivity of this system can be limited. In this study, the Escherichia coli chaperones (GroES-GroEL) were expressed from the PGAP promoter and targeted to the secretory pathway through the endoplasmic reticulum (ER). The ability of the ER targeted chaperones to improve production of bacterial protein in P.pastoris was evaluated. The chaperones tagged with α-factor secretion- and ER retention-signal sequences were co-expressed with either extracellularly secreted phytase or intracellular d-phenylglycine aminotransferase (D-PhgAT) enzymes. The ER residing GroEL-GroES successfully increased the levels of active phytase extracellularly, 1.5-2.3-fold higher than the phytase expression alone, but did not enhance the formation of active, intracellular D-PhgAT. These results indicate that the chaperones have the potential to enhance production of active enzymes when present in the same trafficking pathway. This is the first report on the improvement of extracellular bacterial protein production through co-expression with ER residing bacterial chaperones in the Pichia system. The modified P.pastoris expression system may be beneficial for extracellular expression of other prokaryotic proteins.

A novel bi-directional promoter system allows tunable recombinant protein production in Pichia pastoris

BackgroundThe methylotrophic yeast Pichia pastoris is a well-studied host organism for recombinant protein production, which is usually regulated either by a constitutive promoter (e.g. promoter of glyceraldehyde-3-phosphate dehydrogenase; PGAP) or an inducible promoter (e.g. promoter of alcohol oxidase 1; PAOX1). Both promoter systems have several advantages and disadvantages; with one of the main disadvantages being their lack of tunability. Various novel promoter systems, which are either inducible or de-repressed, allowing higher degrees of freedom, have been reported. Recently, bi-directional promoter systems in P. pastoris with two promoter systems regulating recombinant expression of one or more genes were developed. In this study, we introduce a novel bi-directional promoter system combining a modified catalase promoter system (PDC; derepressible and inducible) and the traditional PAOX1, allowing tunable recombinant protein production.ResultsWe characterized a recombinant P. pastoris strain, carrying the novel bi-directional promoter system, during growth and production in three dynamic bioreactor cultivations. We cloned the model enzyme cellobiohydralase downstream of either promoter and applied different feeding strategies to determine the physiological boundaries of the strain. We succeeded in demonstrating tunability of recombinant protein production solely in response to the different feeding strategies and identified a mixed feed regime allowing highest productivity.ConclusionIn this feasibility study, we present the first controlled bioreactor experiments with a recombinant P. pastoris strain carrying a novel bi-directional promotor combination of a catalase promoter variant (PDC) and the traditional PAOX1. We demonstrated that this bi-directional promoter system allows tunable recombinant protein expression only in response to the available C-sources. This bi-directional promoter system offers a high degree of freedom for bioprocess design and development, making bi-directional promoters in P. pastoris highly attractive for recombinant protein production.

Systems biotechnology for protein production in Pichia pastoris.

The methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) is one of the most important production systems for heterologous proteins. After the first genome sequences were published in 2009, tremendous effort was made to establish systems-level analytical methods. Methylotrophic lifestyle was one of the most thoroughly investigated topics, studied at the levels of transcriptome, proteome and metabolic flux. Also the responses of P. pastoris to environmental stress conditions experienced during high cell density production processes were studied. Metabolomics and flux analysis revealed the plasticity of the cellular metabolism in its adaption to the production of foreign proteins and served as blueprints for subsequent cell engineering and/or process design. The transcriptional response elicited by overexpression of heterologous proteins seems to depend on the nature and complexity of the recombinant product. Based on these data, novel targets for strain engineering could be deduced from transcriptomics and proteomics data mining and effectively enhanced protein secretion. Transcriptional regulation data also served as a valuable resource to identify novel promoters with the desired regulatory characteristics. This review aims to provide a comprehensive overview of systems biology applications in P. pastoris ranging from increased understanding of cell physiology to improving recombinant protein production in this cell factory.

Increasing pentose phosphate pathway flux enhances recombinant protein production in Pichia pastoris.

Production of heterologous proteins in Pichia pastoris (syn. Komagataella sp.) has been shown to exert a metabolic burden on the host metabolism. This burden is associated with metabolite drain, which redirects nucleotides and amino acids from primary metabolism. On the other hand, recombinant protein production affects energy and redox homeostasis of the host cell. In a previous study, we have demonstrated that overexpression of single genes of the oxidative pentose phosphate pathway (PPP) had a positive influence on recombinant production of cytosolic human superoxide dismutase (hSOD). In this study, different combinations of these genes belonging to the oxidative PPP were generated and analyzed. Thereby, a 3.8-fold increase of hSOD production was detected when glucose-6-phosphate dehydrogenase (ZWF1) and 6-gluconolactonase (SOL3) were simultaneously overexpressed, while the combinations of other genes from PPP had no positive effect on protein production. By measuring isotopologue patterns of 13C-labelled metabolites, we could detect an upshift in the flux ratio of PPP to glycolysis upon ZWF1 and SOL3 co-overexpression, as well as increased levels of 6-phosphogluconate. The substantial improvement of hSOD production by ZWF1 and SOL3 co-overexpression appeared to be connected to an increase in PPP flux. In conclusion, we show that overexpression of SOL3 together with ZWF1 enhanced both the PPP flux ratio and hSOD accumulation, providing evidence that in P. pastoris Sol3 limits the flux through PPP and recombinant protein production.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7363-5) contains supplementary material, which is available to authorized users.

A step forward to improve recombinant protein production in Pichia pastoris: From specific growth rate effect on protein secretion to carbon-starving conditions as advanced strategy

The recombinant protein production platform based on the GAP promoter and Pichia pastoris as a host has become a very promising system from an industrial point of view. The need for highly productive bioprocesses gives grounds for the optimization of fermentation strategies maximizing yields and/or productivities, which are often associated with cell growth. Coherent with previous studies, a positive effect of high specific growth rate (μ) on the productivity was observed in carbon-limited chemostat cultivations secreting an antibody fragment. Notably, no significant impact of this factor could be observed in the balance intra- and extracellular of the product. Accordingly, fed-batch cultures operating at a constant high μ were conducted. Furthermore, short carbon-starving periods were introduced along the exponential substrate feeding phase. Strikingly, it was observed an important increase of specific production rate (qP) during such short carbon-starving periods in relation to the exponential substrate feeding intervals. Therefore, the application of carbon-starving periods as an innovative operational strategy was proposed, resulting into increments up to 50% of both yields and total production. The implementation of the proposed substrate feeding profiles should be complementary to cell engineering strategies to improve the relation qP vs μ, thereby enhancing the overall bioprocess efficiency.