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Multiple backbone N-methylation and macrocyclization improve the proteolytic stability and oral availability of therapeutic peptides. Chemical synthesis of such peptides is challenging, in particular for the generation of peptide libraries for screening purposes. Enzymatic backbone N-methylation and macrocyclization occur as part of both non-ribosomal and ribosomal peptide biosynthesis, exemplified by the fungal natural products cyclosporin A and omphalotin A, respectively. Omphalotin A, a 9fold backbone N-methylated dodecamer isolated from the agaricomycete Omphalotus olearius, can be produced in Pichia pastoris by coexpression of the ophMA and ophP genes coding for the peptide precursor protein harbouring an autocatalytic peptide α-N-methyltransferase domain, and a peptide macrocyclase, respectively. Since both OphMA and OphP were previously shown to be relatively promiscuous in terms of peptide substrates, we expressed mutant versions of ophMA, encoding OphMA variants with altered core peptide sequences, along with wildtype ophP and assessed the production of the respective peptide macrocycles by the platform by high-performance liquid chromatography, coupled with tandem mass spectrometry (HPLC-MS/MS). Our results demonstrate the successful production of fifteen non-natural omphalotin-derived macrocycles, containing polar, aromatic and charged residues, and, thus, suggest that the system may be used as biotechnological platform to generate libraries of non-natural multiply backbone N-methylated peptide macrocycles., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The genus Hanseniaspora is characterized by some of the smallest genomes among budding yeasts. These fungi are primarily found on plant surfaces and in fermented products and represent promising biocontrol agents against notorious fungal plant pathogens. In this work, we identify pantothenate auxotrophy of a Hanseniaspora meyeri isolate that shows strong antagonism against the plant pathogen Fusarium oxysporum. Furthermore, strong biocontrol activity in vitro required both pantothenate and biotin in the growth medium. We show that the H. meyeri isolate APC 12.1 can obtain the vitamin from plants and other fungi. The underlying reason for the auxotrophy is the lack of two key pantothenate biosynthesis genes, but six genes encoding putative pantothenate transporters are present in the genome. By constructing and using a Saccharomyces cerevisiae reporter strain, we identified one Hanseniaspora transporter that conferred pantothenate uptake activity to S. cerevisiae. Pantothenate auxotrophy is rare and has been described in only a few bacteria and in S. cerevisiae strains that were isolated from sake. Such auxotrophic strains may seem an unexpected and unlikely choice as potential biocontrol agents, but they may be particularly competitive in their ecological niche and their specific growth requirements are an inherent biocontainment strategy preventing uncontrolled growth in the environment. Auxotrophic strains, such as the H. meyeri isolate APC 12.1, may thus represent a promising strategy for developing biocontrol agents that will be easier to register than prototrophic strains, which are normally used for such applications. IMPORTANCE As a precursor of the essential coenzyme A (CoA), pantothenate is present in all organisms. Plants, bacteria, and fungi are known to synthesize this vitamin, while animals must obtain it through their diet. Pantothenate auxotrophy has not been described in naturally occurring, environmental fungi and is an unexpected property for an antagonistic yeast. Here, we report that yeasts from the genus Hanseniaspora lack key enzymes for pantothenate biosynthesis and identify a transporter responsible for the acquisition of pantothenate from the environment. Hanseniaspora isolates are strong antagonists of fungal plant pathogens. Their pantothenate auxotrophy is a natural biocontainment feature that could make such isolates interesting candidates for new biocontrol approaches and allow easier registration as plant protection agents than prototrophic strains., Competing Interests: The authors declare no conflict of interest.

Saccharomyces cerevisiae cells are capable of responding to mating pheromone only prior to their exit from the G(1) phase of the cell cycle. Ste5 scaffold protein is essential for pheromone response because it couples pheromone receptor stimulation to activation of the appropriate mitogen-activated protein kinase (MAPK) cascade. In naïve cells, Ste5 resides primarily in the nucleus. Upon pheromone treatment, Ste5 is rapidly exported from the nucleus and accumulates at the tip of the mating projection via its association with multiple plasma membrane-localized molecules. We found that concomitant with its nuclear export, the rate of Ste5 turnover is markedly reduced. Preventing nuclear export destabilized Ste5, whereas preventing nuclear entry stabilized Ste5, indicating that Ste5 degradation occurs mainly in the nucleus. This degradation is dependent on ubiquitin and the proteasome. We show that Ste5 ubiquitinylation is mediated by the SCF(Cdc4) ubiquitin ligase and requires phosphorylation by the G(1) cyclin-dependent protein kinase (cdk1). The inability to efficiently degrade Ste5 resulted in pathway activation and cell cycle arrest in the absence of pheromone. These findings reveal that maintenance of this MAPK scaffold at an appropriately low level depends on its compartment-specific and cell cycle-dependent degradation. Overall, this mechanism provides a novel means for helping to prevent inadvertent stimulus-independent activation of a response and for restricting and maximizing the signaling competence of the cell to a specific cell cycle stage, which likely works hand in hand with the demonstrated role that G(1) Cdk1-dependent phosphorylation of Ste5 has in preventing its association with the plasma membrane.

The yeast transcription factor Gcn4p contains two stretches of amino acid residues, NLS1 and NLS2, which are independently able to relocate the cytoplasmic protein chorismate mutase into the nucleus. Only NLS2 is conserved among fungi. A truncated version of CPCA (the counterpart of Gcn4p in Aspergillus nidulans), which lacks the conserved NLS, accumulates in the cytoplasm instead of the nucleus. Nuclear uptake mediated by the NLS1 of Gcn4p is impaired by defects in genes for several different karyopherins, whereas NLS2-dependent nuclear import specifically requires the alpha-importin Srp1p and the beta-importin Kap95p. Yeast strains that are defective in either of these two karyopherins are unable to respond to amino acid starvation. We have thus identified Gcn4p as a substrate for the Srp1p/Kap95p transport complex. Our data suggest that NLS2 is the essential and specific nuclear transport signal; NLS1 may play only an unspecific or accessory role.

Ubiquitin-dependent proteolysis of specific target proteins is required for several important steps during the cell cycle. Degradation of such proteins is strictly cell cycle-regulated and triggered by two large ubiquitin ligases, termed anaphase-promoting complex (APC) and Skp1/Cullin/F-box complex (SCF). Here we show that yeast Ran-binding protein 1 (Yrb1p), a predominantly cytoplasmic protein implicated in nucleocytoplasmic transport, is required for cell cycle regulated protein degradation. Depletion of Yrb1p results in the accumulation of unbudded G(1) cells and of cells arrested in mitosis implying a function of Yrb1p in the G(1)/S transition and in the progression through mitosis. Temperature-sensitive yrb1-51 mutants are defective in APC-mediated degradation of the anaphase inhibitor protein Pds1p and in degradation of the cyclin-dependent kinase inhibitor Sic1p, a target of SCF. Thus, Yrb1p is crucial for efficient APC- and SCF-mediated proteolysis of important cell cycle regulatory proteins. We have identified the UBS1 gene as a multicopy suppressor of yrb1-51 mutants. Ubs1p is a nuclear protein, and its deletion is synthetic lethal with a yrb1-51 mutation. Interestingly, UBS1 was previously identified as a multicopy suppressor of cdc34-2 mutants, which are defective in SCF activity. We suggest that Ubs1p may represent a link between nucleocytoplasmic transport and ubiquitin ligase activity.

The RanGTP-binding protein RanBP1, which is located in the cytoplasm, has been implicated in release of nuclear export complexes from the cytoplasmic side of the nuclear pore complex. Here we show that Yrb1 (the yeast homolog of RanBP1) shuttles between the nucleus and the cytoplasm. Nuclear import of Yrb1 is a facilitated process that requires a short basic sequence within the Ran-binding domain (RBD). By contrast, nuclear export of Yrb1 requires an intact RBD, which forms a ternary complex with the Xpo1 (Crm1) NES receptor in the presence of RanGTP. Nuclear export of Yrb1, however, is insensitive towards leptomycin B, suggesting a novel type of substrate recognition between Yrb1 and Xpo1. Taken together, these data suggest that ongoing nuclear import and export is an important feature of Yrb1 function in vivo.

Saccharomyces cerevisiae Los1p, which is genetically linked to the nuclear pore protein Nsp1p and several tRNA biogenesis factors, was recently grouped into the family of importin/karyopherin-beta-like proteins on the basis of its sequence similarity. In a two-hybrid screen, we identified Nup2p as a nucleoporin interacting with Los1p. Subsequent purification of Los1p from yeast demonstrates its physical association not only with Nup2p but also with Nsp1p. By the use of the Gsp1p-G21V mutant, Los1p was shown to preferentially bind to the GTP-bound form of yeast Ran. Furthermore, overexpression of full-length or N-terminally truncated Los1p was shown to have dominant-negative effects on cell growth and different nuclear export pathways. Finally, Los1p could interact with Gsp1p-GTP, but only in the presence of tRNA, as revealed in an indirect in vitro binding assay. These data confirm the homology between Los1p and the recently identified human exportin for tRNA and reinforce the possibility of a role for Los1p in nuclear export of tRNA in yeast.

CSE1 is essential for yeast cell viability and has been implicated in chromosome segregation. Based on its sequence similarity, Cse1p has been grouped into the family of importin beta-like nucleocytoplasmic transport receptors with highest homology to the recently identified human nuclear export receptor for importin alpha, CAS. We demonstrate here that Cse1p physically interacts with yeast Ran and yeast importin alpha (Srp1p) in the yeast two-hybrid system and that recombinant Cse1p, Srp1p and Ran-GTP form a trimeric complex in vitro. Re-export of Srp1p from the nucleus into the cytoplasm and nuclear uptake of a reporter protein containing a classical NLS are inhibited in a cse1 mutant strain. These findings suggest that Cse1p is the exportin of importin alpha in yeast.

Activity of the tyrosine-inhibitable 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) from Saccharomyces cerevisiae that was encoded by the ARO4 gene cloned on a high-copy-number plasmid was enhanced 64-fold as compared to the wild-type. The enzyme was purified to apparent homogeneity from the strain that harbored this recombinant plasmid. The estimated molecular weight of 42,000 of the enzyme corresponded to the calculated molecular mass of 40 kDa deduced from the DNA sequence. The enzyme could be inactivated by EDTA in a reaction that was reversed by several bivalent metal ions; presumably a metal cofactor is required for enzymatic catalysis. The Michaelis constant of the enzyme was 125 microM for phosphoenolpyruvate and 500 microM for erythrose 4-phosphate. The rate constant was calculated as 6 s-1, and kinetic data indicated a sequential mechanism of the enzymatic reaction. Tyrosine was a competitive inhibitor with phosphoenolpyruvate as substrate of the enzyme (Ki of 0.9 microM) and a noncompetitive inhibitor with erythrose 4-phosphate as substrate. This is in contrast to the ARO3-encoded isoenzyme, where phenylalanine is a competitive inhibitor with erythrose 4-phosphate as a substrate of the enzyme and a noncompetitive inhibitor with phosphoenolpyruvate as substrate.

The HIS7 gene of Saccharomyces cerevisiae encodes a bifunctional glutamine amidotransferase: cyclase that catalyzes the formation of biosynthetic precursors for histidine and adenine. HIS7 is activated by Gcn4p upon amino acid starvation and by the Bas1/2p complex in response to adenine limitation. Mutation analysis of the HIS7 promoter in a deltagcn4 background revealed a polyd(A/T) stretch and a d(CT) repeat as essential elements for Gcn4p-independent basal HIS7 transcription. The protein binding this element was enriched and identified as the multifunctional DNA-binding protein Abf1p. Abf1p binds specifically to the d(CT) repeat sequence, which represents a novel Abf1p-binding motif, and protects 17 nucleotides from digestion by DNase I. In addition, Abf1p binding causes bending of the HIS7 promoter structure.

The number of identified yeast factors involved in transcription has dramatically increased in recent years and the understanding of the interplay between the different factors has become more and more puzzling. Transcription initiation at the core promoter of mRNA encoding genes consisting of upstream, TATA and initiator elements requires an approximately ribosome-sized complex of more than 50 polypeptides. The recent identification and isolation of an RNA polymerase holoenzyme which seems to be preassembled before interacting with a promoter allowed a better understanding of the roles, assignments and interplays of the various constituents of the basal transcription machinery. Recruitment of this complex to the promoter is achieved by numerous interactions with a variety of DNA-bound proteins. These interactions can be direct or mediated by additional adaptor proteins. Other proteins negatively affect transcription by interrupting the recruitment process through protein-protein or protein-DNA interactions. Some basic features of cis-acting elements, the transcriptional apparatus and various trans-acting factors involved in the initiation of mRNA synthesis in yeast are summarized.

Although serine and glycine are ubiquitous amino acids the genetic and biochemical regulation of their synthesis has not been studied in detail. The SER1 gene encodes 3-phosphoserine aminotransferase which catalyzes the formation of phosphoserine from 3-phosphohydroxy-pyruvate, which is obtained by oxidation of 3-phosphoglycerate, an intermediate of glycolysis. Saccharomyces cerevisiae cells provided with fermentable carbon sources mainly use this pathway (glycolytic pathway) to synthesize serine and glycine. We report the isolation of the SER1 gene by complementation and the disruption of the chromosomal locus. Sequence analysis revealed an open reading frame encoding a protein with a predicted molecular weight of 43,401 Da. A previously described mammalian progesterone-induced protein shares 47% similarity with SER1 over the entire protein, indicating a common function for both proteins. We demonstrate that SER1 transcription is regulated by the general control of amino-acid biosynthesis mediated by GCN4. Additionally, DNaseI protection experiments proved the binding of GCN4 protein to the SER1 promoter in vitro and three GCN4 recognition elements (GCREs) were identified. Furthermore, there is evidence for an additional regulation by serine end product repression.

The ARO3 gene of Saccharomyces cerevisiae codes for the phenylalanine-inhibited 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (EC 4.1.2.15) and is regulated by the general control system of amino acid biosynthesis through a single GCN4-binding site in its promoter. A combined deletion and mutation analysis of the ARO3 promoter region in a delta gcn4-background revealed two additional regulatory systems involved in ARO3 transcription. The ARO3 gene is (i) activated through a sequence element which binds the multifunctional DNA-binding protein ABF1 in vitro and (ii) repressed through an URS1 element, which binds the same protein in vitro as the URS1 element in the CAR1 promoter. Since both the ABF1-binding site and the URS1 element represent cis-acting elements of global transcription regulatory systems in yeast, the ARO3 gene is the first example of a GCN4-regulated gene which is both activated and repressed by global transcription factors. Activation of the ARO3 gene through the ABF1-binding site and repression through the URS1 element seem to be independent of each other and independent of activation by the GCN4 protein.

We have fused representatives of three structurally and functionally distinct classes of mammalian transcription activation domains for RNA polymerase II to the yeast GAL4 DNA binding domain. All fusion proteins were stable when expressed in yeast and were tested for their ability to activate transcription from various positions in the yeast GAL1 promoter. Activation domains functional from remote as well as TATA-proximal positions in mammalian cells, e.g. the acidic-type domain of VP16, also stimulate transcription in yeast from various promoter positions. Proline-rich domains, as e.g. in AP-2 and CTF/NF1, with considerable promoter activity and low enhancer activity in mammalian cells stimulate transcription in yeast only from a position close to the TATA box. The glutamine-rich domains of Oct1, Oct2 and Sp1, which activate transcription in mammalian cells from close to the TATA box in response to a remote enhancer, are inactive in the yeast GAL1 promoter. This finding might reflect some basic difference between the organization of yeast and mammalian promoters.

In Saccharomyces cerevisiae, two differently regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (DAHPS; EC 4.1.2.15) isoenzymes carry out the first step in the shikimate pathway. Mutations in both genes are necessary to cause aromatic amino acid (aa) auxotrophy, since one isoenzyme alone is sufficient to produce enough DAHP for normal growth of the cells. The phenylalanine-inhibited DAHPS is encoded by the previously isolated and characterized ARO3 gene. Here, we report the cloning and characterization of the ARO4 gene, encoding the second DAHPS, which is inhibited by tyrosine. The aa sequence of the ARO4 gene product reveals 76% similarity to the ARO3-encoded isoenzyme and 66 and 73% to the three DAHPS isoenzymes from Escherichia coli. ARO4 gene expression is regulated by the general control system of aa biosynthesis. As in the case of the ARO3 gene, a single GCN4-recognition element in the promoter is responsible for derepression of the ARO4 gene under aa starvation conditions. However, in contrast to the situation in the isogene, ARO3, GCN4 does not contribute to the basal level of ARO4 transcription under nonderepressing conditions.

We describe a convenient method for the in vivo construction of large plasmids that possess a multitude of restriction sites. A large (23 kbases) circular self-replicating plasmid carrying a partial LEU2-d gene was cotransformed with a circular non-replicating plasmid carrying the entire LEU2 gene. In vivo recombination results preferentially in a plasmid that carries both the LEU2-d and the entire LEU2 gene. In addition we also found one plasmid with a tandem LEU2 insertion and one plasmid where the LEU2-d gene was replaced by the entire LEU2 gene.