Browse
Submit Data
Databases
API
Help

Dataset Information

27 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker's yeast.

ABSTRACT: Fungi are a valuable source of enzymatic diversity and therapeutic natural products including antibiotics. Here we engineer the baker's yeast Saccharomyces cerevisiae to produce and secrete the antibiotic penicillin, a beta-lactam nonribosomal peptide, by taking genes from a filamentous fungus and directing their efficient expression and subcellular localization. Using synthetic biology tools combined with long-read DNA sequencing, we optimize productivity by 50-fold to produce bioactive yields that allow spent S. cerevisiae growth media to have antibacterial action against Streptococcus bacteria. This work demonstrates that S. cerevisiae can be engineered to perform the complex biosynthesis of multicellular fungi, opening up the possibility of using yeast to accelerate rational engineering of nonribosomal peptide antibiotics.

SUBMITTER: Awan AR

PROVIDER: S-EPMC5418595 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker's yeast.

Awan Ali R AR Blount Benjamin A BA Bell David J DJ Shaw William M WM Ho Jack C H JCH McKiernan Robert M RM Ellis Tom T

Nature communications 20170504

Fungi are a valuable source of enzymatic diversity and therapeutic natural products including antibiotics. Here we engineer the baker's yeast Saccharomyces cerevisiae to produce and secrete the antibiotic penicillin, a beta-lactam nonribosomal peptide, by taking genes from a filamentous fungus and directing their efficient expression and subcellular localization. Using synthetic biology tools combined with long-read DNA sequencing, we optimize productivity by 50-fold to produce bioactive yields ...[more]

PMID: 28469278

Similar Datasets

De novo biosynthesis of vanillin in fission yeast (Schizosaccharomyces pombe) and baker's yeast (Saccharomyces cerevisiae).

Project description:Vanillin is one of the world's most important flavor compounds, with a global market of 180 million dollars. Natural vanillin is derived from the cured seed pods of the vanilla orchid (Vanilla planifolia), but most of the world's vanillin is synthesized from petrochemicals or wood pulp lignins. We have established a true de novo biosynthetic pathway for vanillin production from glucose in Schizosaccharomyces pombe, also known as fission yeast or African beer yeast, as well as in baker's yeast, Saccharomyces cerevisiae. Productivities were 65 and 45 mg/liter, after introduction of three and four heterologous genes, respectively. The engineered pathways involve incorporation of 3-dehydroshikimate dehydratase from the dung mold Podospora pauciseta, an aromatic carboxylic acid reductase (ACAR) from a bacterium of the Nocardia genus, and an O-methyltransferase from Homo sapiens. In S. cerevisiae, the ACAR enzyme required activation by phosphopantetheinylation, and this was achieved by coexpression of a Corynebacterium glutamicum phosphopantetheinyl transferase. Prevention of reduction of vanillin to vanillyl alcohol was achieved by knockout of the host alcohol dehydrogenase ADH6. In S. pombe, the biosynthesis was further improved by introduction of an Arabidopsis thaliana family 1 UDP-glycosyltransferase, converting vanillin into vanillin beta-D-glucoside, which is not toxic to the yeast cells and thus may be accumulated in larger amounts. These de novo pathways represent the first examples of one-cell microbial generation of these valuable compounds from glucose. S. pombe yeast has not previously been metabolically engineered to produce any valuable, industrially scalable, white biotech commodity.

| S-EPMC2681717 | biostudies-literature

Enterotoxicity of a nonribosomal peptide causes antibiotic-associated colitis.

Project description:Antibiotic therapy disrupts the human intestinal microbiota. In some patients rapid overgrowth of the enteric bacterium Klebsiella oxytoca results in antibiotic-associated hemorrhagic colitis (AAHC). We isolated and identified a toxin produced by K. oxytoca as the pyrrolobenzodiazepine tilivalline and demonstrated its causative action in the pathogenesis of colitis in an animal model. Tilivalline induced apoptosis in cultured human cells in vitro and disrupted epithelial barrier function, consistent with the mucosal damage associated with colitis observed in human AAHC and the corresponding animal model. Our findings reveal the presence of pyrrolobenzodiazepines in the intestinal microbiota and provide a mechanism for colitis caused by a resident pathobiont. The data link pyrrolobenzodiazepines to human disease and identify tilivalline as a target for diagnosis and neutralizing strategies in prevention and treatment of colitis.

| S-EPMC4246982 | biostudies-literature

Crosslinking studies of protein-protein interactions in nonribosomal peptide biosynthesis.

Project description:Selective protein-protein interactions between nonribosomal peptide synthetase (NRPS) proteins, governed by communication-mediating (COM) domains, are responsible for proper translocation of biosynthetic intermediates to produce the natural product. In this study, we developed a crosslinking assay, utilizing bioorthogonal probes compatible with carrier protein modification, for probing the protein interactions between COM domains of NRPS enzymes. Employing the Huisgen 1,3-dipolar cycloaddition of azides and alkynes, we examined crosslinking of cognate NRPS modules within the tyrocidine pathway and demonstrated the sensitivity of our panel of crosslinking probes toward the selective protein interactions of compatible COM domains. These studies indicate that copper-free crosslinking substrates uniquely offer a diagnostic probe for protein-protein interactions. Likewise, these crosslinking probes serve as ideal chemical tools for structural studies between NRPS modules where functional assays are lacking.

| S-EPMC2743379 | biostudies-literature

Fungal siderophore biosynthesis catalysed by an iterative nonribosomal peptide synthetase.

Project description:Siderophores play a vital role in the viability of fungi and are essential for the virulence of many pathogenic fungal species. Despite their importance in fungal physiology and pathogenesis, the programming rule of siderophore assembly by fungal nonribosomal peptide synthetases (NRPSs) remains unresolved. Here, we report the characterization of the bimodular fungal NRPS, SidD, responsible for construction of the extracellular siderophore fusarinine C. The use of intact protein mass spectrometry, together with in vitro biochemical assays of native and dissected enzymes, provided snapshots of individual biosynthetic steps during NPRS catalysis. The adenylation and condensation domain of SidD can iteratively load and condense the amino acid building block cis-AMHO, respectively, to synthesize fusarinine C. Our study showcases the iterative programming features of fungal siderophore-producing NRPSs.

| S-EPMC8162485 | biostudies-literature

The Nonribosomal Peptide Valinomycin: From Discovery to Bioactivity and Biosynthesis.

Project description:Valinomycin is a nonribosomal peptide that was discovered from Streptomyces in 1955. Over the past more than six decades, it has received continuous attention due to its special chemical structure and broad biological activities. Although many research papers have been published on valinomycin, there has not yet been a comprehensive review that summarizes the diverse studies ranging from structural characterization, biogenesis, and bioactivity to the identification of biosynthetic gene clusters and heterologous biosynthesis. In this review, we aim to provide an overview of valinomycin to address this gap, covering from 1955 to 2020. First, we introduce the chemical structure of valinomycin together with its chemical properties. Then, we summarize the broad spectrum of bioactivities of valinomycin. Finally, we describe the valinomycin biosynthetic gene cluster and reconstituted biosynthesis of valinomycin. With that, we discuss possible opportunities for the future research and development of valinomycin.

| S-EPMC8068249 | biostudies-literature

A proteomic survey of nonribosomal peptide and polyketide biosynthesis in actinobacteria.

Project description:Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a "protein-first" method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes using mass spectrometry-based proteomics for the targeted detection of expressed nonribosomal peptide synthetases or polyketide synthases. Improvements to the original PrISM screening approach (Nat. Biotechnol. 2009, 27, 951-956), for example, improved de novo peptide sequencing, have enabled the discovery of 10 NRPS/PKS gene clusters from 6 strains. Taking advantage of the concurrence of biosynthetic enzymes and the secondary metabolites they generate, two natural products were associated with their previously "orphan" gene clusters. This work has demonstrated the feasibility of a proteomics-based strategy for use in screening for NRP/PK production in actinomycetes (often >8 Mbp, high GC genomes) versus the bacilli (2-4 Mbp genomes) used previously.

| S-EPMC3253196 | biostudies-literature

The cycloaspeptides: uncovering a new model for methylated nonribosomal peptide biosynthesis.

Project description:The cycloaspeptides are bioactive pentapeptides produced by various filamentous fungi, which have garnered interest from the agricultural industry due to the reported insecticidal activity of the minor metabolite, cycloaspeptide E. Genome sequencing, bioinformatics and heterologous expression confirmed that the cycloaspeptide gene cluster contains a minimal 5-module nonribosomal peptide synthetase (NRPS) and a new type of trans-acting N-methyltransferase (N-MeT). Deletion of the N-MeT encoding gene and subsequent feeding studies determined that two modules of the NRPS preferentially accept and incorporate N-methylated amino acids. This discovery allowed the development of a system with unprecedented control over substrate supply and thus output, both increasing yields of specific metabolites and allowing the production of novel fluorinated analogues. Furthermore, the biosynthetic pathway to ditryptophenaline, another fungal nonribosomal peptide, was shown to be similar, in that methylated phenylalanine is accepted by the ditryptophenaline NRPS. Again, this allowed the directed biosynthesis of a fluorinated analogue, through the feeding of a mutant strain. These discoveries represent a new paradigm for the production of N-methylated cyclic peptides via the selective incorporation of N-methylated free amino acids.

| S-EPMC5941284 | biostudies-literature

Engineering and elucidation of the lipoinitiation process in nonribosomal peptide biosynthesis.

Project description:Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but the lack of an in-depth understanding of starter condensation domain-mediated lipoinitiation limits the bioengineering of NRPSs to obtain novel derivatives with desired acyl chains. Here, we show that the acyl chains of the lipopeptides rhizomide, holrhizin, and glidobactin were modified by engineering the starter condensation domain, suggesting a workable approach to change the acyl chain. Based on the structure of the mutated starter condensation domain of rhizomide biosynthetic enzyme RzmA in complex with octanoyl-CoA and related point mutation experiments, we identify a set of residues responsible for the selectivity of substrate acyl chains and extend the acyl chains from acetyl to palmitoyl. Furthermore, we illustrate three possible conformational states of starter condensation domains during the reaction cycle of the lipoinitiation process. Our studies provide further insights into the mechanism of lipoinitiation and the engineering of nonribosomal peptide synthetases.

| S-EPMC7804268 | biostudies-literature

Discovery and Biosynthesis of Azabicyclene, a Conserved Nonribosomal Peptide in Pseudomonas aeruginosa.

Project description:Azabicyclene, an azetidine-containing natural product, was identified using quorum-sensing molecules to upregulate expression of a gene cluster highly conserved in the human pathogen Pseudomonas aeruginosa. Mutational studies of the gene cluster revealed essential genes for biosynthesis, including an unusual nonribosomal peptide synthetase. Reconstitution of this enzyme in vitro identified key biosynthetic intermediates. This work demonstrates a useful strategy for discovering quorum-sensing-regulated natural products. It sets the stage for understanding the biosynthesis and bioactivity of azabicyclene.

| S-EPMC7179086 | biostudies-literature

Multiple alternate transcripts direct the biosynthesis of microcystin, a cyanobacterial nonribosomal peptide.

Project description:The mcyABCDEFGHIJ gene cluster of Microcystis aeruginosa encodes the mixed polyketide synthase/nonribosomal peptide synthetase (microcystin synthetase) which is responsible for biosynthesis of the potent liver toxin microcystin. The sequence and orientation of the mcy genes have previously been reported, but no transcriptional analysis had been performed prior to this study. The mcyABCDEFGHIJ genes are transcribed as two polycistronic operons, mcyABC and mcyDEFGHIJ, from a central bidirectional promoter between mcyA and mcyD. Two transcription start sites were detected for both mcyA and mcyD when cells were exposed to light intensities of 68 and 16 micromol of photons m(-2) s(-1). The start sites, located 206 and 254 bp upstream of the translational start for mcyD under high and low light conditions, respectively, indicate long untranslated leader regions. Putative transcription start sites were also identified for mcyE, mcyF, mcyG, mcyH, mcyI, and mcyJ but not for mcyB and mcyC. A combination of reverse transcription-PCR and rapid amplification of cDNA ends was employed throughout this work, which may have been one of the first transcriptional analyses of a large nonribosomal polyketide gene cluster.

| S-EPMC126702 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data