Pushing and pulling proteins into the yeast secretory pathway enhances recombinant protein secretion

Yeasts and especially Pichia pastoris (syn Komagataella spp.) are popular microbial expression systems for the production of recombinant proteins. One of the key advantages of yeast host systems is their ability to secrete the recombinant protein into the culture media. However, secretion of some recombinant proteins is less efficient. These proteins include antibody fragments such as Fabs or scFvs. We have recently identified translocation of nascent Fab fragments from the cytosol into the endoplasmic reticulum (ER) as one major bottleneck. Conceptually, this bottleneck requires engineering to increase the flux of recombinant proteins at the translocation step by pushing on the cytosolic side and pulling on the ER side. This engineering strategy is well-known in the field of metabolic engineering. To apply the push-and-pull strategy to recombinant protein secretion, we chose to modulate the cytosolic and ER Hsp70 cycles, which have a key impact on the translocation process. After identifying the relevant candidate factors of the Hsp70 cycles, we combined the push-and-pull factors in a single strain and achieved synergistic effects for antibody fragment secretion. With this concept we were able to successfully engineer strains and improve protein secretion up to 5-fold for different model protein classes. Overall, titers of more than 1.3 g/L Fab and scFv were reached in bioreactor cultivations.     

Impaired Cutinase Secretion in Saccharomyces cerevisiae Induces Irregular Endoplasmic Reticulum (ER) Membrane Proliferation, Oxidative Stress, and ER-Associated Degradation

Impaired secretion of the hydrophobic CY028 cutinase invokes an unfolded protein response (UPR) in Saccharomyces cerevisiae cells. Here we show that the UPR in CY028-expressing S. cerevisiae cells is manifested as an aberrant morphology of the endoplasmic reticulum (ER) and as extensive membrane proliferation compared to the ER morphology and membrane proliferation of wild-type CY000-producing S. cerevisiae cells. In addition, we observed oxidative stress, which resulted in a 21-fold increase in carbonylated proteins in the CY028-producing S. cerevisiae cells. Moreover, CY028-producing S. cerevisiae cells use proteasomal degradation to reduce the amount of accumulated CY028 cutinase, thereby attenuating the stress invoked by CY028 cutinase expression. This proteasomal degradation occurs within minutes and is characteristic of ER-associated degradation (ERAD). Our results clearly show that impaired secretion of the heterologous, hydrophobic CY028 cutinase in S. cerevisiae cells leads to protein aggregation in the ER, aberrant ER morphology and proliferation, and oxidative stress, as well as a UPR and ERAD.     

Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells

Almost all of the 200 or so approved biopharmaceuticals have been produced in one of three host systems: the bacterium Escherichia coli, yeasts (Saccharomyces cerevisiae, Pichia pastoris) and mammalian cells. We describe the most widely used methods for the expression of recombinant proteins in the cytoplasm or periplasm of E. coli, as well as strategies for secreting the product to the growth medium. Recombinant expression in E. coli influences the cell physiology and triggers a stress response, which has to be considered in process development. Increased expression of a functional protein can be achieved by optimizing the gene, plasmid, host cell, and fermentation process. Relevant properties of two yeast expression systems, S. cerevisiae and P. pastoris, are summarized. Optimization of expression in S. cerevisiae has focused mainly on increasing the secretion, which is otherwise limiting. P. pastoris was recently approved as a host for biopharmaceutical production for the first time. It enables high-level protein production and secretion. Additionally, genetic engineering has resulted in its ability to produce recombinant proteins with humanized glycosylation patterns. Several mammalian cell lines of either rodent or human origin are also used in biopharmaceutical production. Optimization of their expression has focused on clonal selection, interference with epigenetic factors and genetic engineering. Systemic optimization approaches are applied to all cell expression systems. They feature parallel high-throughput techniques, such as DNA microarray, next-generation sequencing and proteomics, and enable simultaneous monitoring of multiple parameters. Systemic approaches, together with technological advances such as disposable bioreactors and microbioreactors, are expected to lead to increased quality and quantity of biopharmaceuticals, as well as to reduced product development times.     

Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii

To explore the potential of Ashbya gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4-methylumbelliferyl-β-d-lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae.     

Pharmaceutical protein production by yeast: towards production of human blood proteins by microbial fermentation

1. Download : Download high-res image (259KB)
2. Download : Download full-size image

Highlights? Recombinant therapeutic protein production is a multibillion dollar market. ? E. coli comprises 30% of recombinant protein production but not suitable for human therapeutics. ? Eukaryotic systems other than yeast are costly or not so efficient regarding protein yields. ? S. cerevisiae shows a high potential to be a suitable platform for therapeutic proteins. ? Human blood proteins are the next candidates to be challenged by S. cerevisiae system.     

Yeast Secretes High Amounts of Human Calreticulin without Cellular Stress

The ER chaperone calreticulin (CALR) also has extracellular functions and can exit the mammalian cell in response to various factors, although the mechanism by which this takes place is unknown. The yeast Saccharomyces cerevisiae efficiently secretes human CALR, and the analysis of this process in yeast could help to clarify how it gets out of eukaryotic cells. We have achieved a secretion titer of about 140 mg/L CALR in our S. cerevisiae system. Here, we present a comparative quantitative whole proteome study in CALR-secreting yeast using non-equilibrium pH gradient electrophoresis (NEPHGE)-based two-dimensional gel electrophoresis (2DE) as well as liquid chromatography mass spectrometry in data-independent analysis mode (LC-MS^E). A reconstructed carrier ampholyte (CA) composition of NEPHGE-based first-dimension separation for 2DE could be used instead of formerly commercially available gels. Using LC-MS^E, we identified 1574 proteins, 20 of which exhibited differential expression. The largest group of differentially expressed proteins were structural ribosomal proteins involved in translation. Interestingly, we did not find any signs of cellular stress which is usually observed in recombinant protein-producing yeast, and we did not identify any secretory pathway proteins that exhibited changes in expression. Taken together, high-level secretion of human recombinant CALR protein in S. cerevisiae does not induce cellular stress and does not burden the cellular secretory machinery. There are only small changes in the cellular proteome of yeast secreting CALR at a high level.     

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

Background

Pichia pastoris is widely used as a production platform for heterologous proteins and model organism for organelle proliferation. Without a published genome sequence available, strain and process development relied mainly on analogies to other, well studied yeasts like Saccharomyces cerevisiae.

Results

To investigate specific features of growth and protein secretion, we have sequenced the 9.4 Mb genome of the type strain DSMZ 70382 and analyzed the secretome and the sugar transporters. The computationally predicted secretome consists of 88 ORFs. When grown on glucose, only 20 proteins were actually secreted at detectable levels. These data highlight one major feature of P. pastoris, namely the low contamination of heterologous proteins with host cell protein, when applying glucose based expression systems. Putative sugar transporters were identified and compared to those of related yeast species. The genome comprises 2 homologs to S. cerevisiae low affinity transporters and 2 to high affinity transporters of other Crabtree negative yeasts. Contrary to other yeasts, P. pastoris possesses 4 H⁺/glycerol transporters.

Conclusion

This work highlights significant advantages of using the P. pastoris system with glucose based expression and fermentation strategies. As only few proteins and no proteases are actually secreted on glucose, it becomes evident that cell lysis is the relevant cause of proteolytic degradation of secreted proteins. The endowment with hexose transporters, dominantly of the high affinity type, limits glucose uptake rates and thus overflow metabolism as observed in S. cerevisiae. The presence of 4 genes for glycerol transporters explains the high specific growth rates on this substrate and underlines the suitability of a glycerol/glucose based fermentation strategy. Furthermore, we present an open access web based genome browser http://www.pichiagenome.org.     

Quorum sensing-mediated protein degradation for dynamic metabolic pathway control in Saccharomyces cerevisiae

Dynamic regulation has been widely applied to optimize metabolic flux distribution. However, compared with prokaryotes, quorum sensing-mediated pathway control is still very limited in Saccharomyces cerevisiae. In this study, we designed quorum sensing-regulated protein degradation circuits for dynamic metabolic pathway control in S. cerevisiae. The synthetic quorum sensing circuits were developed by integration of a plant hormone cytokinin system with the endogenous yeast Ypd1-Skn7 signal transduction pathway and the positive feedback circuits were optimized by promoter engineering. We then constructed an auxin-inducible protein degradation system and used quorum sensing circuits to regulate auxin synthesis to achieve dynamic control of protein degradation. As a demonstration, the circuits were applied to control Erg9 degradation to produce α-farnesene and the titer of α-farnesene increased by 80%. The population-regulated protein degradation system developed here extends dynamic regulation to the protein level in S. cerevisiae and is a promising approach for metabolic pathway control.     

Avoiding entry into intracellular protein degradation pathways by signal mutations increases protein secretion in Pichia pastoris

In our previous study, we serendipitously discovered that protein secretion in the methylotrophic yeast Pichia pastoris is enhanced by a mutation (V50A) in the mating factor alpha (MFα) prepro-leader signal derived from Saccharomyces cerevisiae. In the present study, we investigated 20 single-amino-acid substitutions, including V50A, located within the MFα signal peptide, indicating that V50A and several single mutations alone provided significant increase in production of the secreted proteins. In addition to hydrophobicity index analysis, both an unfolded protein response (UPR) biosensor analysis and a microscopic observation showed a clear difference on the levels of UPR induction and mis-sorting of secretory protein into vacuoles among the wild-type and mutated MFα signal peptides. This work demonstrates the importance of avoiding entry of secretory proteins into the intracellular protein degradation pathways, an observation that is expected to contribute to the engineering of strains with increased production of recombinant secreted proteins.     

The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57–70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future.     

Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins

One of the most important branches of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems are used for the production of recombinant proteins including bacteria, yeasts, molds, mammals, plants, and insects. Yeast expression systems such as Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris) are more popular. P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology. Overall, the benefits of protein production by P. pastoris system include appropriate folding (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins to the external environment of the cell. Moreover, in the P. pastoris expression system due to its limited production of endogenous secretory proteins, the purification of recombinant protein is easy. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Although P. pastoris expression systems are impressive and easy to use with well-defined process protocols, some degree of process optimization is required to achieve maximum production of the target proteins. Methanol and sorbitol concentration, Mut forms, temperature and incubation time have to be adjusted to obtain optimal conditions, which might vary among different strains and externally expressed protein. Eventually, optimal conditions for the production of a recombinant protein in P. pastoris expression system differ according to the target protein.     

Expression of bacterial xylose isomerase in Saccharomyces cerevisiae under galactose supplemented condition

We constructed recombinant Saccharomyces cerevisiae harboring the xylose isomerase (XI) gene isolated from Clostridium phytofermentans to metabolize xylose and use it as a carbon and energy source. In this study, the effect of supplementation using co-substrate such as glucose or galactose on xylose utilization was studied in recombinant S. cerevisiae. Glucose, which is transported with high affinity by the same transport system as is xylose, was not affected by the heterologous expression of XI, thus xylose utilization was not observed in recombinant S. cerevisiae. However, supplemental galactose added to the recombinant S. cerevisiae stimulated xylose utilization as well as the expression of XI protein. Recombinant S. cerevisiae consumed up to 23.48 g/L of xylose when grown in media containing 40 g/L of xylose and supplemented with 20 g/L of galactose. These cells also produced 15.89 g/L of ethanol. Therefore, expression of the bacterial XI in recombinant S. cerevisiae was highly induced by the addition of supplemental galactose as a co-substrate with xylose, and supplemented galactose enabled the yeast strain to grow on xylose and ferment xylose to ethanol.     

Evaluating fermentation effects on cell growth and crude extract metabolic activity for improved yeast cell-free protein synthesis

Saccharomyces cerevisiae is a promising source organism for the development of a practical, eukaryotic crude extract based cell-free protein synthesis (CFPS) system. Crude extract CFPS systems represent a snapshot of the active metabolism in vivo, in response to the growth environment at the time of harvest. Therefore, fermentation plays a central role in determining metabolic activity in vitro. Here, we developed a fermentation protocol using chemically defined media to maximize extract performance for S. cerevisiae-based CFPS. Using this new protocol, we obtained a 4-fold increase in protein synthesis yields with extracts derived from wild-type S288c as compared to a previously developed protocol that uses complex growth media. The final luciferase yield in our new method was 8.86 ± 0.28 μg mL⁻¹ in a 4 h batch reaction. For each of the extracts processed under different fermentation conditions, synthesized protein, precursor monomers (amino acids), and energy substrates (nucleotides) were evaluated to analyze the effect of the changes in the growth environment on cell-free metabolism. This study underscores the critical role fermentation plays in preparing crude extract for CFPS reactions and offers a simple strategy to regulate desired metabolic activity for cell-free synthetic biology applications based on crude cell extracts.     

Heterologous protein secretion by Candida utilis

The yeast Candida utilis (also referred to as Torula) is used as a whole-cell food additive and as a recombinant host for production of intracellular molecules. Here, we report recombinant C. utilis strains secreting significant amounts of Candida antarctica lipase B (CalB). Native and heterologous secretion signals led to secretion of CalB into the growth medium; CalB was enzymatically active and it carried a short N-glycosyl chain lacking extensive mannosylation. Furthermore, CalB fusions to the C. utilis Gas1 cell wall protein led to effective surface display of enzymatically active CalB and of β-galactosidase. Secretory production in C. utilis was achieved using a novel set of expression vectors containing sat1 conferring nourseothricin resistance, which could be transformed into C. utilis, Pichia jadinii, Candida albicans, and Saccharomyces cerevisiae; C. utilis promoters including the constitutive TDH3 and the highly xylose-inducible GXS1 promoters allowed efficient gene expression. These results establish C. utilis as a promising host for the secretory production of proteins.     

Comparison of two expression platforms in respect to protein yield and quality: Pichia pastoris versus Pichia angusta

The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor (MTF). Both recombinant proteins were engineered to contain an N-terminal Strep- (WSHPQFEK) and a C-terminal His₆-tag. In addition, both proteins contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion. Following vector construction, transformation and zeocin amplification, the best Pichia producers were identified in a screening procedure using Western blot and a Luminex xMAP™ based high-throughput method. Recombinant NK1-fragment and MTF were purified from culture supernatants of the best producers by affinity chromatography (Ni–nitrilotriacetic acid columns). Using P. pastoris as a host for the synthesis of NK1-fragment a protein yield of 5.7 mg/l was achieved. In comparable expression experiments P. angusta yielded 1.6 mg/l of NK1-fragment. NK1-fragment apparently was not glycosylated in either system. For the production of MTF, P. pastoris was also the superior host yielding 1.2 mg/l glycosylated recombinant protein whereas P. angusta was clearly less efficient (<0.2 mg/l MTF). For both expression systems no correlation between the amount of recombinant protein and the copy number of the chromosomally integrated heterologous genes was found. In P. pastoris strains less degradation of the two model recombinant proteins was observed. Altogether, this paper provides a structured protocol for rapidly identifying productive Pichia strains for the synthesis of full-length recombinant proteins.     

Extracellular recombinant protein production under continuous culture conditions with Escherichia coli using an alternative plasmid selection mechanism

The secretion of recombinant proteins into the extracellular space by Escherichia coli presents advantages like easier purification and protection from proteolytic degradation. The controlled co-expression of a bacteriocin release protein aids in moving periplasmic proteins through the outer membrane. Since such systems have rarely been applied in continuous culture it seemed to be attractive to study the interplay between growth-phase regulated promoters controlling release protein genes and the productivity of a chemostat process. To avoid the use of antibiotics and render this process more sustainable, alternative plasmid selection mechanisms were required. In the current study, the strain E. coli JM109 harboring plasmid p582 was shown to stably express and secrete recombinant β-glucanase in continuous culture using a minimal medium. The segregational instability of the plasmid in the absence of antibiotic selection pressure was demonstrated. The leuB gene, crucial in the leucine biosynthetic pathway, was cloned onto plasmid p582 and the new construct transformed into an E. coli Keio (ΔleuB) knockout strain. The ability of the construct to complement the leucine auxotrophy was initially tested in shake-flasks and batch cultivation. Later, this strain was successfully grown for more than 200 h in a chemostat and was found to be able to express the recombinant protein. Significantly, it showed a stable maintenance of the recombinant plasmid in the absence of any antibiotics. The plasmid stability in a continuously cultivated E. coli fermentation, in the absence of antibiotics, with extracellular secretion of recombinant protein provides an interesting model for further improvements.     

Inhibition of protein secretion and protein kinase activity in the locust fat body by diamide (Azodicarboxylic acid-bis-dimethylamide)

Protein secretion from locust fat body incubated in vitro is instantaneously halted in the presence of diamide (azodicarboxylic acid-bis-dimethylamide). Independent inhibition of amino acid uptake, protein synthesis (verified in cell-free systems), and protein secretion is demonstrated. Diamide inhibits cyclic AMP- and cyclic GMP-dependent protein kinase activity in locust fat body.     

A study of uptake of radiolabeled host proteins and protein synthesis during development of eggs of the endoparasitoid,Microplitis croceipes (Cresson) (Braconidae)

The uptake of radiolabeled haemolymph and fat body proteins from fourth instar larvae of Heliothis zea (Boddie) by eggs of Microplitis croceipes (Cresson) was examined by SDS-polyacrylamide gel electrophoresis and by autoradiography. None of the ¹²⁵I-labeled haemolymph proteins was detected in eggs exposed to the proteins in vivo. Although several of the proteins were observed in eggs incubated with the labeled proteins in vitro, none of these proteins was degraded or resynthesized into new structural proteins during development of the embryo. Similarly, no significant uptake of labeled fat body proteins by the eggs could be detected in vitro. On the other hand, protein synthesis measured by incorporation of [³⁵S]methionine occurred throughout egg development. Proteins were synthesized at least 1 hr after the egg was deposited into the host. The protein patterns of eggs on one-dimensional SDS gels were complex and ranged in size from less than 18,500 to more than 330,000 mol. wt. The protein band patterns of the newly synthesized proteins showed some qualitative differences at 1–8, 16–32 and 40 hr after egg deposition. We conclude that eggs do not absorb or utilize the host apoproteins (or degradation products) but instead synthesize proteins de novo from free amino acids in the host haemolymph.