Saccharomyces cerevisiaeCells Harboring the Gene Encoding Sarcotoxin IA Secrete a Peptide That Is Toxic to Plant Pathogenic Bacteria (original) (raw)
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Journal of Peptide Science, 2008
New methods of safe biological pest control are required as a result of evolution of insect resistance to current biopesticides. Yeast strains being developed for conversion of cellulosic biomass to ethanol are potential host systems for expression of commercially valuable peptides, such as bioinsecticides, to increase the cost-effectiveness of the process. Spider venom is one of many potential sources of novel insect-specific peptide toxins. Libraries of mutants of the small amphipathic peptide lycotoxin-1 from the wolf spider were produced in high throughput using an automated integrated plasmid-based functional proteomic platform and screened for ability to kill fall armyworms, a significant cause of damage to corn (maize) and other crops in the United States. Using amino acid scanning mutagenesis (AASM) we generated a library of mutagenized lycotoxin-1 open reading frames (ORF) in a novel small ubiquitin-like modifier (SUMO) yeast expression system. The SUMO technology enhanced expression and improved generation of active lycotoxins. The mutants were engineered to be expressed at high level inside the yeast and ingested by the insect before being cleaved to the active form (so-called Trojan horse strategy). These yeast strains expressing mutant toxin ORFs were also carrying the xylose isomerase (XI) gene and were capable of aerobic growth on xylose. Yeast cultures expressing the peptide toxins were prepared and fed to armyworm larvae to identify the mutant toxins with greatest lethality. The most lethal mutations appeared to increase the ability of the toxin α-helix to interact with insect cell membranes or to increase its pore-forming ability, leading to cell lysis. The toxin peptides have potential as value-added coproducts to increase the cost-effectiveness of fuel ethanol bioproduction.
Protein expression and …, 1998
or signal sequence, which promotes their translocation a-Sarcin is a ribosome-inactivating protein from into the rough endoplasmic reticulum (1). These signal the mold Aspergillus giganteus. The methylotrophic sequences show a number of conserved features that yeast Pichia pastoris has been transformed with two are essential for protein export (2), although displaying plasmids (pHILD2preaS and pHILS1preaS), which different interactions with the translocation machinery contain the complete a-sarcin cDNA, including its (3). The late secretory steps involve specific proteolysis original fungal leader peptide, under the control of of the proproteins to yield the corresponding secreted yeast alcohol oxidase promoter. The second one is mature forms, Kex2p protease being the best known indeed fused to the signal sequence of P. pastoris member of a family of homologous eukaryotic endopepacid phosphatase. The transformed yeasts secreted tidases involved in such steps (4). both mature and pro-a-sarcin. The presence of this a-Sarcin is a cytotoxic protein, active against several pro-a-sarcin in the yeast extracellular medium is due human tumor cell lines (5), secreted by the mold Asperto an inefficient recognition of the pro-sequence by a gillus giganteus MDH 18894 (6). This cytotoxin belongs putative Kex2p-like endopeptidase. A third plasmid to a family of highly similar proteins from different accounting for a single mutation of the a-sarcin Aspergillus spp. (restrictocin, mitogillin, AspF1, clavin, leader peptide was designed to produce a more effiand gigantin). Their cytotoxicity is due to their ribocient Kex2p recognition motif. This approach renucleolytic activity against a single phosphodiester sulted in the extracellular production of only the mabond of the larger rRNA, resulting in the so-called ature protein, suggesting the existence of a two-step fragment of about 400 nt length (7), which leads to mechanism for processing its leader peptide. This recombinant a-sarcin is identical to the original fungal inhibition of protein biosynthesis (8). a-Sarcin is able protein, according to activity and spectroscopic cri-to hydrolyze this bond in ribosomes from all eukaryotes teria. In addition, pro-a-sarcin, which has been charand prokaryotes tested so far (9-12) because the region acterized for the first time, also exhibits ribonucleoaround the cleavage site is evolutionary conserved. lytic activity as the mature protein does. Therefore, Therefore, a-sarcin could be used as a good model for protection of the producing cells against this kind of protein secretion, since inefficient export of this protein ribotoxins may depend on an efficient recognition of would potentially lead to cell death. In this context, the the signal sequence followed by translocation of the posttranslational processing, to render extracellular nascent polypeptide to the endoplasmic reticulum. mature toxin, and the self-protection of the toxin-pro-᭧ 1998 Academic Press ducing ribosomes are intriguing questions for this kind of cytotoxins. Although the sar gene has been cloned and efficiently expressed in Escherichia coli (13), a host Secreted eukaryotic proteins are synthesized as preeukaryote system needs to be used to answer these proproteins, with an NH 2-terminal targeting domain, questions. Restrictocin, from Aspergillus restrictus, has been produced in A.
MGG - Molecular & General Genetics, 1997
The Aspergillus niger and Trichoderma reesei genes encoding the functional homologues of the small GTP-binding protein SAR1p, which is involved in the secretion pathway in Saccharomyces cerevisiae, have been cloned and characterised. The A. niger gene (sarA) contains ®ve introns, whereas the T. reesei gene (sar1) has only four. In both cases the ®rst intron is at the same position as the single S. cerevisiae SAR1 intron. The encoded proteins show 70±80% identity to the SAR1 protein. Complementation of S. cerevisiae sar1 and sec12 mutants by expression vectors carrying the A. niger sarA and T. reesei sar1 cDNA clones con®rmed that the cloned genes are functional homologues of the S. cerevisiae SAR1 gene. Three mutant alleles of the A. niger sarA gene (D29G, E109K, D29G/E109K), generated by site-directed mutagenesis, revealed a thermosensitive dominant-negative phenotype in the presence of the wild-type sarA allele. This result contrasts with the situation in S. cerevisiae, where similar mutations have a thermosensitive phenotype. Taken together, our results indicate that the sarA gene is involved in an essential function in A. niger.
Production of recombinant sarcotoxin IA in Bombyx mori cells
The Biochemical journal, 1990
A cDNA for sarcotoxin IA, an antibacterial protein of Sarcophaga peregrina (fleshfly), was inserted into a silkworm baculovirus vector and expressed in Bm-N cells, a line of Bombyx mori cells. When a cysteine proteinase inhibitor, p-chloromercuribenzenesulphonic acid, was present in the culture medium, a significant amount of recombinant sarcotoxin IA accumulated, but without this reagent the product seemed to be degraded in this system. The C-terminus of the recombinant sarcotoxin IA seemed to be glycine, not amidated arginine as found in authentic sarcotoxin IA. Probably, Bm-N cells lack the C-terminal alpha-amidation enzyme.
A peptide from insects protects transgenic tobacco from a parasitic weed
Transgenic Research, 2005
Parasitic plants present some of the most intractable weed problems for agriculture in much of the world. Species of root parasites such as Orobanche can cause enormous yield losses, yet few control measures are effective and affordable. An ideal solution to this problem is the development of parasite-resistant crops, but this goal has been elusive for most susceptible crops. Here we report a mechanism for resistance to the parasitic angiosperm Orobanche based on expression of sarcotoxin IA in transgenic tobacco. Sarcotoxin IA is a 40-residue peptide with antibiotic activity, originally isolated from the fly, Sarcophaga peregrina. The sarcotoxin IA gene was fused to an Orobanche-inducible promoter, HMG2, which is induced locally in the host root at the point of contact with the parasite, and used to transform tobacco. The resulting transgenic plants accumulated more biomass than non-transformed plants in the presence of parasites. Furthermore, plants expressing sarcotoxin IA showed enhanced resistance to O. aegyptiaca as evidenced by abnormal parasite development and higher parasite mortality after attachment as compared to non-transformed plants. The transgenic plants were similar in appearance to non-transformed plants suggesting that sarcotoxin IA is not detrimental to the host.
Yeast killer toxins: from ecological significance to application
Critical Reviews in Biotechnology, 2019
Killer toxins are proteins that are often glycosylated and bind to specific receptors on the surface of their target microorganism, which is then killed through a target-specific mode of action. The killer phenotype is widespread among yeast and about 100 yeast killer species have been described to date. The spectrum of action of the killer toxins they produce targets spoilage and pathogenic microorganisms. Thus, they have potential as natural antimicrobials in food and for biological control of plant pathogens, as well as therapeutic agents against animal and human infections. In spite of this wide range of possible applications, their exploitation on the industrial level is still in its infancy. Here, we initially briefly report on the biodiversity of killer toxins and the ecological significance of their production. Their actual and possible applications in the agrofood industry are discussed, together with recent advances in their heterologous production and the manipulation for development of peptide-based therapeutic agents.
Mapping of functional domains within the Saccharomyces cerevisiae type 1 killer preprotoxin
EMBO Journal, 1986
Strains of Saccharomyces cerevisiae harboring M1-dsRNA, the determinant of type 1 killer and immunity phenotypes, secrete a dimeric 19-kd toxin that kills sensitive yeast cells by the production of cation-permeable pores in the cytoplasmic membrane. The preprotoxin, an intracellular precursor to toxin, has the domain sequence delta-alpha-gamma-beta where alpha and beta are the 9.5-and 9.0-kd subunits of secreted toxin. Plasmids containing a partial cDNA copy of M1, in which alpha, gamma, and beta are fused to the PH05 promoter and signal peptide, have previously been shown to express phosphate-repressible toxin production and immunity. Here the construction of a complete DNA copy of the preprotoxin gene and its mutagenesis are described. Analysis of the expression of these mutants from the PH05 promoter elucidates the functions of the preprotoxin domains. delta acts as a leader peptide and efficiently mediates the secretion, glycosylation and maturation of killer toxin. Mutations wi...
Applied and …, 2011
Wine Saccharomyces cerevisiae strains producing a new killer toxin (Klus) were isolated. They killed all the previously known S. cerevisiae killer strains, in addition to other yeast species, including Kluyveromyces lactis and Candida albicans. The Klus phenotype is conferred by a medium-size double-stranded RNA (dsRNA) virus, Saccharomyces cerevisiae virus Mlus (ScV-Mlus), whose genome size ranged from 2.1 to 2.3 kb. ScV-Mlus depends on ScV-L-A for stable maintenance and replication. We cloned and sequenced Mlus. Its genome structure is similar to that of M1, M2, or M28 dsRNA, with a 5-terminal coding region followed by two internal A-rich sequences and a 3-terminal region without coding capacity. Mlus positive strands carry cis-acting signals at their 5 and 3 termini for transcription and replication similar to those of killer viruses. The open reading frame (ORF) at the 5 portion codes for a putative preprotoxin with an N-terminal secretion signal, potential Kex2p/Kexlp processing sites, and N-glycosylation sites. No sequence homology was found either between the Mlus dsRNA and M1, M2, or M28 dsRNA or between Klus and the K1, K2, or K28 toxin. The Klus amino acid sequence, however, showed a significant degree of conservation with that of the product of the host chromosomally encoded ORF YFR020W of unknown function, thus suggesting an evolutionary relationship. Saccharomyces cerevisiae killer strains produce and secrete protein toxins that are lethal to sensitive strains of the same or related yeast species. These toxins have been grouped into three types, K1, K2, or K28, based on their killing profiles and lack of cross-immunity. Members of each group can kill nonkiller yeasts as well as killer yeasts belonging to the other types. They are immune, however, to their own toxin or to toxins produced by strains of the same killer type (for reviews, see references 21, 32, 33, and 47). K1, K2, and K28 killer toxins are genetically encoded by medium-size double-stranded RNA (dsRNA) viruses grouped into three types, M1, M2, and M28, of 1.6, 1.5, and 1.8 kb, respectively. Only one strand (the positive strand) has coding capacity. In each case, the 5Ј-end region contains an open reading frame (ORF) that codes for the toxin precursor, or preprotoxin (pptox), which also provides immunity. The three toxin-coding M dsRNAs show no sequence homology to each other (35). M viruses depend on a second large (4.6-kb
Protein Express Purif, 1998
α-Sarcin is a ribosome-inactivating protein from the moldAspergillus giganteus.The methylotrophic yeastPichia pastorishas been transformed with two plasmids (pHILD2preαS and pHILS1preαS), which contain the complete α-sarcin cDNA, including its original fungal leader peptide, under the control of yeast alcohol oxidase promoter. The second one is indeed fused to the signal sequence ofP. pastorisacid phosphatase. The transformed yeasts secreted both mature and pro-α-sarcin. The presence of this pro-α-sarcin in the yeast extracellular medium is due to an inefficient recognition of the pro-sequence by a putative Kex2p-like endopeptidase. A third plasmid accounting for a single mutation of the α-sarcin leader peptide was designed to produce a more efficient Kex2p recognition motif. This approach resulted in the extracellular production of only the mature protein, suggesting the existence of a two-step mechanism for processing its leader peptide. This recombinant α-sarcin is identical to the original fungal protein, according to activity and spectroscopic criteria. In addition, pro-α-sarcin, which has been characterized for the first time, also exhibits ribonucleolytic activity as the mature protein does. Therefore, protection of the producing cells against this kind of ribotoxins may depend on an efficient recognition of the signal sequence followed by translocation of the nascent polypeptide to the endoplasmic reticulum.