Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains.

Heterologous protein expression levels in Saccharomyces cerevisiae fermentations are highly dependent on the susceptibility to endogenous yeast proteases. Small peptides, such as glucagon and glucagon-like-peptides (GLP-1 and GLP-2), featuring an open structure are particularly accessible for proteolytic degradation during fermentation. Therefore, homogeneous products cannot be obtained. The most sensitive residues are found at basic amino acid residues in the peptide sequence. These heterologous peptides are degraded mainly by the YPS1-encoded aspartic protease, yapsin1, when produced in the yeast. In this article, distinct degradation products were analyzed by HPLC and mass spectrometry, and high yield of the heterologous peptide production has been achieved by the disruption of the YPS1 gene (previously called YAP3). By this technique, high yield continuous fermentation of glucagon in S. cerevisiae is now possible.

Purification and characterisation of a new hypothalamic satiety peptide, cocaine and amphetamine regulated transcript (CART), produced in yeast.

Cocaine and amphetamine regulated transcript (CART) is a newly discovered hypothalamic peptide with a potent appetite suppressing activity following intracerebroventricular administration. When the mature rat CART sequence encoding CART(1-102) was inserted in the yeast expression plasmid three CART peptides could be purified from the fermentation broth reflecting processing at dibasic sequences. None of these corresponded to the naturally occurring CART(55-102). In order to obtain CART(55-102) the precursor Glu-Glu-Ile-Asp-CART(55-102) has been produced and CART(55-102) was generated by digestion of the precursor with dipeptidylaminopeptidase-1. All four generated CART peptides have been characterised by N-terminal amino acid sequencing and mass spectrometry. The CART peptides contain six cysteine residues and using the yeast expressed CART(62-102) the disulphide bond configuration was found to be I-III, II-V and IV-VI. When the four CART peptides were intracerebroventricularly injected in fasted mice (0.1 to 2.0 microg) they all produced a dose dependent inhibition of food intake.

alpha-Factor pro-peptide N-linked oligosaccharides facilitate secretion of the insulin precursor in Saccharomyces cerevisiae.

To evaluate the possible relationship between N-linked glycosylation of the Saccharomyces cerevisiae alpha-factor pro-peptide and transport of the alpha-factor pro-peptide/insulin precursor fusion protein through the Saccharomyces cerevisiae secretory pathway, we analysed secretion of insulin precursor facilitated by alpha-factor pro-peptides with one or more of the three N-linked glycosylation sites removed. Mutation of the three alpha-factor pro-peptide N-linked glycosylation sites drastically decreased insulin precursor secretion. The three alpha-factor pro-peptide N-linked glycosylation sites differ in their ability to facilitate secretion of the insulin precursor. The two alpha-factor pro-peptide N-linked glycosylation sites localized closest to the insulin precursor contributed significantly to secretion, whereas the most N-terminally linked glycosylation site did not appear to facilitate secretion. Only correctly folded insulin precursor was found in the culture supernatant, regardless of the pro-peptide used for secretion, indicating that alpha-factor pro-peptide N-linked oligosaccharide chains are not necessary for correct folding of the insulin precursor. Thus, N-linked glycosylation facilitates intracellular transport of the alpha-factor propeptide/insulin precursor fusion protein through the Saccharomyces cerevisiae secretory pathway and secretion of the insulin precursor. N-linked glycosylation per se is not sufficient to facilitate secretion of the insulin precursor; the position of the N-linked oligosaccharide chain on the alpha-factor pro-peptide is important for facilitating efficient secretion.

Yeast cell permeabilizing beta-1,3-glucanases: A tool for the integration of downstream processes and metabolic engineering applications to yeast.

In this article, we consider the impact on downstream process design resulting from the use of metabolically engineered yeast strains. We address the issue of how manipulation of cell wall permeability can improve the release and subsequent recovery of heterologous products produced in yeast.

Synthetic leaders with potential BiP binding mediate high-yield secretion of correctly folded insulin precursors from Saccharomyces cerevisiae.

Secretion leaders are essential for expression of many heterologous proteins including insulin in yeast. The function of secretion leaders and their interaction with the secretory pathway is not clear. To determine what constitutes functional pre-pro-leader sequences in Saccharomyces cerevisiae, synthetic leader sequences for secretion of the insulin precursor were developed by a combination of rational design and stepwise systematic optimization. The synthetic leaders efficiently facilitate secretion of the insulin precursor from S. cerevisiae when compared with the alpha-factor leader, leading to a high yield of correctly folded insulin precursor in the culture supernatant. The synthetic leaders feature two potential N-linked glycosylation sites which are efficiently glycosylated during secretion. Pulse-chase analysis indicates that the synthetic leaders/insulin precursor fusion protein have a prolonged residence in the endoplasmic reticulum compared to the alpha-factor leader/insulin precursor fusion protein. The longer transition time in the endoplasmic reticulum mediated by the synthetic leaders might provide additional time for correct folding of the insulin precursor and account for the increased fermentation yield.

Nucleotide sequence of a beta-1,3-glucanase isoenzyme IIA gene of Oerskovia xanthineolytica LL G109 (Cellulomonas cellulans) and initial characterization of the recombinant enzyme expressed in Bacillus subtilis.

The nucleotide sequence of the betaglIIA gene, encoding the extracellular beta-1,3-glucanase IIA (betaglIIA) of the yeast-lytic actinomycete Oerskovia xanthineolytica LL G109, was determined. Sequence comparison shows that the betaglIIA enzyme has over 80% identity to the betaglII isoenzyme, an endo-beta-1,3-glucanase having low yeast-lytic activity secreted by the same bacterium. The betaglIIA enzyme lacks a glucan- or mannan-binding domain, such as those observed in beta-1,3-glucanases and proteases having high yeast/fungus-lytic activity. It can be included in the glycosyl hydrolase family 16. Gene fusion expression in Bacillus subtilis DN1885 followed by preliminary characterization of the recombinant gene product indicates that betaglIIA has a pI of 3.8 to 4.0 and is active on both laminarin and curdlan, having an acid optimum pH activity (ca. 4.0).

Molecular cloning of a lytic beta-1,3-glucanase gene from Oerskovia xanthineolytica LLG109. A beta-1,3-glucanase able to selectively permeabilize the yeast cell wall.

Molecular cloning of the beta gIII gene encoding for an endo-beta-1,3-glucanase (beta gl II) from Oerskovia xanthineolytica LLG109, a yeast-lytic gram-positive bacterium, has been conducted in order to elucidate its primary sequence and subsequently express it into B. subtilis. This endo-beta-1,3-glucanase exhibits low yeast-lytic activity toward viable S. cerevisiae cells, and it has shown ability to selectively permeabilize the yeast cell wall and release intracellular proteins produced by yeast. Highly degenerate oligonucleotides have been used to PCR-amplify a region of the beta-1,3-glucanase II encoding gene from O. xanthineolytica LLG109. The amplified fragment has been cloned and sequenced. The deduced amino acid sequence contains regions identical to the amino acid sequences previously determined by direct sequencing of the purified enzyme from O. xanthineolytica LLG109. By using the 180-bp PCR product as a homologous probe, we have been able to isolate four positive clones harboring plasmids pPF1A, pPF1B, pPF8A, and pPF9A, respectively, from a partial genomic library from O. xanthineolytica LLG109. All four plasmids contained a 2.7-kb BamHI insert that hybridized to the PCR probe under high stringency conditions. The 2.7-kb fragment seemed to be identical in all four cases regarding preliminary partial restriction mapping analysis done on the four plasmids. The 1.5-kb BamHI/KpnI restriction fragment from pPF8A and pPF9A hybridizing with the 180-bp PCR probe is presently being sequenced. The cloning of the lytic beta-1,3-glucanase from O. xanthineolytica LLG109 expands the number of yeast lytic beta-glucanases so far cloned. The availability of the nucleotide sequences of such a family of genes will allow further understanding of the role and mode of action of these enzymes in yeast cell wall degradation. In addition, a more extensive study on the structure and functional relationships of these enzymes will allow us to engineer "tailor-made" lytic beta-1,3-glucanases for use in new and improved large-scale selective cell permeabilization (SCP) and selective protein recovery (SPR) from yeast cells, not only from S. cerevisiae but also from alternative yeast expression systems such as Hansenula polymorpha, Pichia pastoris, and others, which are becoming of increasing importance in biotechnology.

Aprotinin and aprotinin analogues expressed in yeast.

Synthetic genes encoding aprotinin and aprotinin analogues were constructed and fused in frame to the S. cerevisiae mating factor alpha 1 signal-leader (1-85) sequence. Expression in yeast resulted in secretion into the culture medium of a moderate yield of correctly processed aprotinin (1-58) together with two N-terminally extended forms. Des-Arg1, Pro2-aprotinin was expressed in a higher yield. In this case only the correct N-terminal amino acid sequence was found. Substitution of Ser42 for Arg42 in the potential internal KEX2 processing site improved the secretion yield. The aprotinins are characterized by an inhibition profile similar to that of native aprotinin. Des-Arg1, Pro2-[Arg15, Ser42] aprotinin has a strongly increased plasma kallikrein inhibition profile.

Soluble, prolonged-acting insulin derivatives. III. Degree of protraction, crystallizability and chemical stability of insulins substituted in positions A21, B13, B23, B27 and B30.

It was previously demonstrated that insulins to which positive charge has been added by substituting B13 glutamic acid with a glutamine residue, B27 threonine with an arginine or lysine residue, and by blocking the C-terminal carboxyl group of the B-chain by amidation, featured a prolonged absorption from the subcutis of rabbits and pigs after injection in solution at acidic pH. The phenomenon is ascribed to a low solubility combined with the readiness by which these analogs crystallize as the injectant is being neutralized in the tissue. However, acid solutions of insulin are chemically unstable as A21 asparagine both deamidates to aspartic acid and takes part in formation of covalent dimers via alpha-amino groups of other molecules. In order to circumvent the instability, substitutions were introduced in position A21, in addition to those in B13, B27 and B30, challenging the fact that A21 asparagine has been conserved in this position throughout the evolution. Biological potency was retained when glycine, serine, threonine, aspartic acid, histidine and arginine were introduced in this position, although to a varying degree. In the crystal structure of insulin a hydrogen bond bridges the alpha-nitrogen of A21 with the backbone carbonyl of B23 glycine. In order to investigate the importance of this hydrogen bond for biological activity a gene for the single-chain precursor B-chain(1-29)-Ala-Ala-Lys-A-chain(1-21) featuring an A21 proline was synthesized. However, this single-chain precursor failed to be properly produced by yeast, pointing to the formation of this hydrogen bond as an essential step in the folding process. The stability of the A21-substituted analogs in acid solutions (pH 3-4) with respect to deamidation and formation of dimers was approximately 5-10 times higher than that of human insulin in neutral solution. The rate of absorption of most insulins is decreased by increasing the Zn2+ concentration of the preparation. However, one analog with A21 glycine showed first-order absorption kinetics in pigs with a half-life of approximately 25 h, independent of the Zn2+ concentration. The day-to-day variation of the absorption of this analog was significantly lower than that of the conventional insulin suspensions, a property that might render such an insulin useful in the attempts to improve glucose control in diabetics by a more predictable delivery of basal insulin.

Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30.

It has previously been found that insulins, to which positive charge has been added by substitutions in position B30, thus raising the isoelectric point towards pH 7, had a prolonged action when injected as slightly acidic solutions because such derivatives crystallize very readily upon neutralization. Positive charge has now been added by substituting the B13 and A17 glutamic acid residues with glutamines and B27 threonine with lysine or arginine. These substitutions were introduced by site-specific mutagenesis in a gene coding for a single-chain insulin precursor. By tryptic transpeptidation the single-chain precursors were transformed to the double-chain insulin structure, concomitantly with incorporation of residue B30. Thus insulins combining B13 glutamine, A17 glutamine and B27 lysine or arginine with B30 threonine, threonine amide or lysine amide were synthesized. The time course of blood glucose lowering effect and the absorption were studied after subcutaneous injection in rabbits and pigs. The prolonged action of B30-substituted insulins was markedly enhanced by B27 lysine or arginine substitutions and by B13 glutamine. The B27 residue is located on the surface of the hexamer, so a basic residue in this position presumably promotes the packing of hexamers at neutral pH. The B13 residues cluster in the centre of the hexamer. When the electrostatic repulsive forces from six glutamic acid residues are abolished by substitution with glutamine, a stabilization of the hexamer can be envisaged.(ABSTRACT TRUNCATED AT 250 WORDS)