Introduction of an N-glycosylation site increases secretion of heterologous proteins in yeasts

Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama V(HH) antibody fragments by S. cerevisiae or Pichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion.     

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.     

Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k_cat, the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.     

Expression and Secretion of Defined Cutinase Variants by Aspergillus awamori

Several cutinase variants derived by molecular modelling and site-directed mutagenesis of a cutinase gene from Fusarium solani pisi are poorly secreted by Saccharomyces cerevisiae. The majority of these variants are successfully produced by the filamentous fungus Aspergillus awamori. However, the L51S and T179Y mutations caused reductions in the levels of extracellular production of two cutinase variants by A. awamori. Metabolic labelling studies were performed to analyze the bottleneck in enzyme production by the fungus in detail. These studies showed that because of the single L51S substitution, rapid extracellular degradation of cutinase occurred. The T179Y substitution did not result in enhanced sensitivity towards extracellular proteases. Presumably, the delay in the extracellular accumulation of this cutinase variant is caused by the enhanced hydrophobicity of the molecule. Overexpression of the A. awamori gene encoding the chaperone BiP in the cutinase-producing A. awamori strains had no significant effect on the secretion efficiency of the cutinases. A cutinase variant with the amino acid changes G28A, A85F, V184I, A185L, and L189F that was known to aggregate in the endoplasmic reticulum of S. cerevisiae, resulting in low extracellular protein levels, was successfully produced by A. awamori. An initial bottleneck in secretion occurred before or during translocation into the endoplasmic reticulum but was rapidly overcome by the fungus.     

Impaired Secretion of a Hydrophobic Cutinase by Saccharomyces cerevisiae Correlates with an Increased Association with Immunoglobulin Heavy-Chain Binding Protein (BiP)

This study focuses on the different efficiencies of secretion of two fungal cutinases by Saccharomyces cerevisiae, a wild-type cutinase (CY000) and a hydrophobic mutant cutinase (CY028). Both cutinases are placed under control of the GAL7 promoter, by which the expression levels can be regulated. Wild-type cutinase was secreted at up to 25 mg per g (dry weight), while CY028 was secreted at a level of 2 mg per g (dry weight); this difference is nearly independent of the expression level. Pulse-chase experiments revealed that whereas CY000 cutinase is secreted, CY028 is irreversibly retained in the cell. Immunogold labelling followed by electron microscopy revealed colocalization of CY028 with immunoglobulin heavy-chain binding protein (BiP) in the endoplasmic reticulum (ER). The increase of wild-type cutinase expression did not result in higher levels of the molecular chaperone BiP, but BiP levels are raised by increased induction of the hydrophobic mutant cutinase. Immunoprecipitation studies showed that in contrast to the wild-type cutinase, the hydrophobic mutant cutinase interacts with BiP. These results indicate that the introduction of two exposed hydrophobic patches in cutinase results in a higher affinity for BiP which might cause the retention of this mutant cutinase in the ER.     

High-level expression and characterization of Fusarium solani cutinase in Pichia pastoris

High-level extracellular production of Fusarium solani cutinase was achieved using a Pichia pastoris expression system. The cutinase-encoding gene was cloned into pPICZαA with the Saccharomyces cerevisiae α-factor signal sequence and methanol-inducible alcohol oxidase promoter by two different ways. The additional sequences of the c-myc epitope and (His)₆-tag of the vector were fused to the C-terminus of cutinase, while the other expression vector was constructed without any additional sequence. P. pastoris expressing the non-tagged cutinase exhibited about two- and threefold higher values of protein amount and cutinase activity in the culture supernatant, respectively. After simple purification by diafiltration process, both cutinases were much the same in the specific activity and the biochemical properties such as the substrate specificity and the effects of temperature and pH. In conclusion, the high-level secretion of F. solani cutinase in P. pastoris was demonstrated for the first time and would be a promising alternative to many expression systems previously used for the large-scale production of F. solani cutinase in Saccharomyces cerevisiae as well as Escherichia coli.     

Improvement of Sec-dependent secretion of a heterologous model protein in Bacillus subtilis by saturation mutagenesis of the N-domain of the AmyE signal peptide

Due to the lack of an outer membrane, Gram-positive bacteria (e.g., Bacillus species) are considered as promising host organisms for the secretory production of biotechnologically relevant heterologous proteins. However, the yields of the desired target proteins were often reported to be disappointingly low. Here, we used saturation mutagenesis of the positively charged N-domain (positions 2–7) of the signal peptide of the Bacillus subtilis α-amylase (AmyE) as a novel approach for the improvement of the secretion of a heterologous model protein, cutinase from Fusarium solani pisi, by the general secretory pathway of B. subtilis. Automated high-throughput screening of the resulting signal peptide libraries allowed for the identification of four single point mutations that resulted in significantly increased cutinase amounts, three of which surprisingly reduced the net charge of the N-domain from +3 to +2. Characterization of the effects of the identified mutations on protein synthesis and export kinetics by pulse-chase analyses indicates that an optimal balance between biosynthesis and the flow of the target protein through all stages of the B. subtilis secretion pathway is of crucial importance with respect to yield and quality of secreted heterologous proteins.     

Evaluation and application of constitutive promoters for cutinase production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cutinase as a promising biocatalyst has been intensively studied and applied in processes targeted for industrial scale. In this work, the cutinase gene tfu from Thermobifida fusca was artificially synthesized according to codon usage bias of Saccharomyces cerevisiae and investigated in Saccharomyces cerevisiae. Using the α-factor signal peptide, the T. fusca cutinase was successfully overexpressed and secreted with the GAL1 expression system. To increase the cutinase level and overcome some of the drawbacks of induction, four different strong promoters (ADH1, HXT1, TEF1, and TDH3) were comparatively evaluated for cutinase production. By comparison, promoter TEF1 exhibited an outstanding property and significantly increased the expression level. By fed-batch fermentation with a constant feeding approach, the activity of cutinase was increased to 29.7 U/ml. The result will contribute to apply constitutive promoter TEF1 as a tool for targeted cutinase production in S. cerevisiae cell factory.     

The use of inulinase pre-pro leader peptide for secretion of heterologous proteins in Saccharomyces cerevisiae

Summary The pre-pro and pre regions of inulinase leader peptide have been used to secrete the heterologous proteins such as human lipocortin-1 and human interleukin-2 (IL2) in Saccharomyces cerevisiae. The inulinase leader peptide consisting of both pre and pro regions directed an efficient secretion of such heterologous proteins in S. cerevisiae, while deletion of pro region prevents them from being secreted. Both pre and pro regions of inulinase leader peptide appears to be necessary to direct secretion of heterologous proteins in S. cerevisiae.     

Combinatorial impact of Sec signal peptides from Bacillus subtilis and bioprocess conditions on heterologous cutinase secretion by Corynebacterium glutamicum

The impact of Sec signal peptides (SPs) from Bacillus subtilis in combination with isopropyl-β- d -1-thiogalactopyranoside concentration and feeding profile was investigated for heterologous protein secretion performance by Corynebacterium glutamicum using cutinase as model enzyme. Based on a comprehensive data set of about 150 bench-scale bioreactor cultivations in fed-batch mode and choosing the cutinase yield as objective, it was shown that relative secretion performance for bioprocesses remains very similar, irrespective of the applied SP enabling Sec-mediated cutinase secretion. However, to achieve the maximal absolute cutinase yield, careful adjustment of bioprocess conditions was found to be necessary. A model-based, two-step multiple regression approach resembled the collected data in a comprehensive way. The corresponding results suggest that the choice of the heterologous Sec SP and its interaction with the adjusted exponential feeding profile is highly relevant to maximize absolute cutinase yield in this study. For example, the impact of Sec SP is high at low growth rates and low at high growth rates. However, promising Sec SPs could be inferred from less complex batch cultivations. The extensive data were also evaluated in terms of cutinase productivity, highlighting the well-known trade-off between yield and productivity in bioprocess development in detail. Conclusively, only the right combination of target protein, Sec SP, and bioprocess conditions is the key to success.     

Fatty acid production from butter using novel cutinase-displaying yeast

We used cutinase from the filamentous fungi Aspergillus oryzae to produce dairy flavors. Secretory and displayed forms of cutinase were investigated using salt-free butter, which is composed mostly of triacylglycerides, as the substrate. The secretory form of cutinase, which was produced in recombinant A. oryzae, was suitable for producing butyric acids (16.8 mol%). Also, cutinase displayed on the cell surface of the yeast Saccharomyces cerevisiae as a fusion protein with α-agglutinin released butyric acid at a 2.7-fold rate (45.4 mol%) higher than that of the secreted form. Yeasts carrying two copies of cutinase genes into their chromosomes, which were constructed using the HELOH method, released free fatty acids rapidly and showed 2-fold higher lipase activity compared with yeasts carrying one copy of the cutinase gene.     

Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in Lactococcus lactis

Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in Lactococcus lactis using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SP_Nuc) by that of L. lactis protein Usp45 (SP_Usp) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SP_Usp than when directed by SP_Nuc. This SP_Usp effect on Nuc secretion is not due to a better antifolding activity, since SP_Usp:Nuc precursor proteins display enzymatic activity in vitro, while SP_Nuc:Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895–1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SP_Usp and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis.     

A novel fusion partner for enhanced secretion of recombinant proteins in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Expressing proteins with fusion partners improves yield and simplifies the purification process. We developed a novel fusion partner to improve the secretion of heterologous proteins that are otherwise poorly excreted in yeast. The VOA1 (YGR106C) gene of Saccharomyces cerevisiae encodes a subunit of vacuolar ATPase. We found that C-terminally truncated Voa1p was highly secreted into the culture medium, even when fused with rarely secreted heterologous proteins such as human interleukin-2 (hIL-2). Deletion mapping of C-terminally truncated Voa1p, identified a hydrophilic 28-amino acid peptide (HL peptide) that was responsible for the enhanced secretion of target protein. A purification tag and a protease cleavage site were added to use HL peptide as a multi-purpose fusion partner. The utility of this system was tested via the expression and purification of various heterologous proteins. In many cases, the yield of target proteins fused with the peptide was significantly increased, and fusion proteins could be directly purified with affinity chromatography. The fusion partner was removed by in vitro processing, and intact proteins were purified by re-application of samples to affinity chromatography.     

Extraction of peptide tagged cutinase in detergent-based aqueous two-phase systems

Detergent-based aqueous two-phase systems have the advantage to require only one auxiliary chemical to induce phase separation above the cloud point. In a systematic study the efficiency of tryptophan-rich peptide tags was investigated to enhance the partitioning of an enzyme to the detergent-rich phase using cutinase as an example. Up to 90% enzyme activity could be extracted in a single step from whole broth of recombinant Saccharomyces cerevisiae expressing cutinase variants carrying a (WP)₄ tag. In contrast, the extraction yield of wild type cutinase was 2–3% only. The detergent concentration and the temperature are the main parameters to optimize the extraction yield. Considering availability, extraction yields, and price the detergent Agrimul NRE 1205 served best for enzyme recovery.     

A dual approach for improving homogeneity of a human-type N-glycan structure in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

N-glycosylation is an important feature of therapeutic and other industrially relevant proteins, and engineering of the N-glycosylation pathway provides opportunities for developing alternative, non-mammalian glycoprotein expression systems. Among yeasts, Saccharomyces cerevisiae is the most established host organism used in therapeutic protein production and therefore an interesting host for glycoengineering. In this work, we present further improvements in the humanization of the N-glycans in a recently developed S. cerevisiae strain. In this strain, a tailored trimannosyl lipid-linked oligosaccharide is formed and transferred to the protein, followed by complex-type glycan formation by Golgi apparatus-targeted human N-acetylglucosamine transferases. We improved the glycan pattern of the glycoengineered strain both in terms of glycoform homogeneity and the efficiency of complex-type glycosylation. Most of the interfering structures present in the glycoengineered strain were eliminated by deletion of the MNN1 gene. The relative abundance of the complex-type target glycan was increased by the expression of a UDP-N-acetylglucosamine transporter from Kluyveromyces lactis, indicating that the import of UDP-N-acetylglucosamine into the Golgi apparatus is a limiting factor for efficient complex-type N-glycosylation in S. cerevisiae. By a combination of the MNN1 deletion and the expression of a UDP-N-acetylglucosamine transporter, a strain forming complex-type glycans with a significantly improved homogeneity was obtained. Our results represent a further step towards obtaining humanized glycoproteins with a high homogeneity in S. cerevisiae.     

Reduced Proteolysis of Secreted Gelatin and Yps1-Mediated α-Factor Leader Processing in a Pichia pastoris kex2 Disruptant

Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae α-factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the YPS1 gene had no effect on gelatin degradation or processing of the α-factor propeptide. When both the KEX2 and YPS1 genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that P. pastoris kex2 and yps1 single-gene disruptants can be used for the α-factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.     

A genetic approach for analyzing the co-operative function of the tRNA mimicry complex,eRF1/eRF3, in translation termination on the ribosome

During termination of translation in eukaryotes, a GTP-binding protein, eRF3, functions within a complex with the tRNA-mimicking protein, eRF1, to decode stop codons. It remains unclear how the tRNA-mimicking protein co-operates with the GTPase and with the functional sites on the ribosome. In order to elucidate the molecular characteristics of tRNA-mimicking proteins involved in stop codon decoding, we have devised a heterologous genetic system in Saccharomyces cerevisiae. We found that eRF3 from Pneumocystis carinii (Pc-eRF3) did not complement depletion of S. cerevisiae eRF3. The strength of Pc-eRF3 binding to Sc-eRF1 depends on the GTP-binding domain, suggesting that defects of the GTPase switch in the heterologous complex causes the observed lethality. We isolated mutants of Pc-eRF3 and Sc-eRF1 that restore cell growth in the presence of Pc-eRF3 as the sole source of eRF3. Mapping of these mutations onto the latest 3D-complex structure revealed that they were located in the binding-interface region between eRF1 and eRF3, as well as in the ribosomal functional sites. Intriguingly, a novel functional site was revealed adjacent to the decoding site of eRF1, on the tip domain that mimics the tRNA anticodon loop. This novel domain likely participates in codon recognition, coupled with the GTPase function.     

Mutant strains of Pichia pastoris with enhanced secretion of recombinant proteins

Although Pichia pastoris is a popular protein expression system, it exhibits limitations in its ability to secrete heterologous proteins. Therefore, a REMI (restriction enzyme mediated insertion) strategy was utilized to select mutant beta-g alactosidase s upersecretion (bgs) strains that secreted increased levels of a β-galactosidase reporter. Many of the twelve BGS genes may have functions in intracellular signaling or vesicle transport. Several of these strains also appeared to contain a more permeable cell wall. Preliminary characterization of four bgs mutants showed that they differed in the ability to enhance the export of other reporter proteins. bgs13, which has a disruption in a gene homologous to Saccharomyces cerevisiae protein kinase C (PKC1), gave enhanced secretion of most recombinant proteins that were tested, raising the possibility that it has the universal super-secreter phenotype needed in an industrial production strain of P. pastoris.