Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker’s yeast

Awan, Ali Raza; Blount, Benjamin A.; Bell, David; Shaw, W. M.; Ho, Jack C H; McKiernan, Robert M; Ellis, Tom

doi:10.1038/ncomms15202

Cited by 84 publications

(81 citation statements)

References 31 publications

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“…Several beneficial effects of compartmentalization are known and have been summarized in Table 1. Heterologous expression of the penicillin biosynthetic genes in S. cerevisiae, has been achieved [9,37,96]. Very low concentrations of penicillin were detected in the S. cerevisiae culture broth indicating that the yeast compartmentalization and secretion system is not optimal for biosynthesis of this metabolite.…”

Section: Benefits Of Subcellular Compartmentalization and Difficultiementioning

confidence: 99%

Transport systems, intracellular traffic of intermediates and secretion of β-lactam antibiotics in fungi

Martı́n

2020

Fungal Biol Biotechnol

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Fungal secondary metabolites are synthesized by complex biosynthetic pathways catalized by enzymes located in different subcellular compartments, thus requiring traffic of precursors and intermediates between them. The β-lactam antibiotics penicillin and cephalosporin C serve as an excellent model to understand the molecular mechanisms that control the subcellular localization of secondary metabolites biosynthetic enzymes. Optimal functioning of the β-lactam biosynthetic enzymes relies on a sophisticated temporal and spatial organization of the enzymes, the intermediates and the final products. The first and second enzymes of the penicillin pathway, ACV synthetase and IPN synthase, in Penicillium chrysogenum and Aspergillus nidulans are cytosolic. In contrast, the last two enzymes of the penicillin pathway, phenylacetyl-CoA ligase and isopenicillin N acyltransferase, are located in peroxisomes working as a tandem at their optimal pH that coincides with the peroxisomes pH. Two MFS transporters, PenM and PaaT have been found to be involved in the import of the intermediates isopenicillin N and phenylacetic acid, respectively, into peroxisomes. Similar compartmentalization of intermediates occurs in Acremonium chrysogenum; two enzymes isopenicillin N-CoA ligase and isopenicillin N-CoA epimerase, that catalyse the conversion of isopenicillin N in penicillin N, are located in peroxisomes. Two genes encoding MFS transporters, cefP and cefM, are located in the early cephalosporin gene cluster. These transporters have been localized in peroxisomes by confocal fluorescence microscopy. A third gene of A. chrysogenum, cefT, encodes an MFS protein, located in the cell membrane involved in the secretion of cephalosporin C, although cefT-disrupted mutants are still able to export cephalosporin by redundant transporters. The secretion of penicillin from peroxisomes to the extracellular medium is still unclear. Attempts have been made to identify a gene encoding the penicillin secretion protein among the 48 ABC-transporters of P. chrysogenum. The highly efficient secretion system that exports penicillin against a concentration gradient may involve active penicillin extrusion systems mediated by vesicles that fuse to the cell membrane. However, there is no correlation of pexophagy with penicillin or cephalosporin formation since inactivation of pexophagy leads to increased penicillin or cephalosporin biosynthesis due to preservation of peroxisomes. The penicillin biosynthesis finding shows that in order to increase biosynthesis of novel secondary metabolites it is essential to adequately target enzymes to organelles.

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Section: Benefits Of Subcellular Compartmentalization and Difficultiementioning

confidence: 99%

Transport systems, intracellular traffic of intermediates and secretion of β-lactam antibiotics in fungi

Martı́n

2020

Fungal Biol Biotechnol

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“…These microbes have already been exploited for the biosynthesis of high‐value natural products—therefore, synthesis of antibiotics is a logical next step . A notable recent study by Awan et al attempted biosynthesis of penicillin G (benzylpenicillin) in baker's yeast ( Saccharomyces cerevisiae ) . Though antimicrobial resistance has decreased the usage of this previous mainstay antibiotic, it is still used in treatment and is produced in vast quantities (60 000 tons per year) .…”

Section: Chassismentioning

confidence: 99%

“…Approaches used for the expression of antibiotic BGCs in non‐Streptomyces organisms. Methods include combinatorial DNA assembly of benzylpenicillin in Saccharomyces cerevisiae , Red/ET recombineering, and TAR cloning in Bacillus subtilis . In the shown example of combinatorial assembly, a library of pathway genes attached to promoters of varying strength is generated.…”

Section: Chassismentioning

confidence: 99%

The “Three Cs” of Novel Antibiotic Discovery and Production through Synthetic Biology: Biosynthetic Gene Clusters, Heterologous Chassis, and Synthetic Microbial Consortia

2018

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Antimicrobial resistance is a rapidly growing problem worldwide, with bacteria showing resistance to last‐resort antibiotic treatments such as carbapenems and colistin becoming more abundant. Synthetic biology may be able to contribute to solving this growing antimicrobial resistance crisis by facilitating the engineering of diversified collections of novel antibiotics, targeting multidrug resistant bacteria in new ways. This review discusses recent advances in the use of synthetic biology methodologies to discover and produce novel antimicrobials; in particular, the work being carried out on biosynthetic gene clusters, heterologous chassis, and synthetic microbial consortia, the “three Cs” of the title.

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“…Engineered compartmentalization has garnered considerable attention in recent years 7,[14][15][16][17][18] .…”

Section: Introductionmentioning

confidence: 99%

Repurposing the yeast peroxisome to compartmentalize a toxic enzyme enables improved (S)-reticuline production

Grewal¹,

Samson²,

Baker

et al. 2020

Preprint

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Eukaryotic cells compartmentalize metabolic pathways in organelles to achieve optimal reaction conditions and avoid crosstalk with other factors in the cytosol. Increasingly, engineers are researching ways in which synthetic compartmentalization could be used to address challenges in metabolic engineering. Here, we identified that norcoclaurine synthase (NCS), the enzyme which catalyzes the first committed reaction in benzylisoquinoline alkaloid (BIA) biosynthesis, is toxic when expressed cytosolically in Saccharomyces cerevisiae and, consequently, restricts (S)-reticuline production. We developed a compartmentalization strategy that alleviates NCS toxicity while promoting increased (S)-reticuline titer, achieved through efficient targeting of toxic NCS to the peroxisome while, crucially, taking advantage of the free flow of metabolite substrates and product across the peroxisome membrane. We identified that peroxisome protein capacity in S. cerevisiae becomes a limiting factor for further improvement of BIA production and demonstrate that expression of engineered transcription factors can mimic the oleate response for larger peroxisomes, further increasing BIA titer without the requirement for peroxisome induction with fatty acids. This work specifically addresses the challenges associated with toxic NCS expression and, more broadly, highlights the potential for engineering organelles with desired characteristics for metabolic engineering.

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Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker’s yeast

Cited by 84 publications

References 31 publications

Transport systems, intracellular traffic of intermediates and secretion of β-lactam antibiotics in fungi

Transport systems, intracellular traffic of intermediates and secretion of β-lactam antibiotics in fungi

The “Three Cs” of Novel Antibiotic Discovery and Production through Synthetic Biology: Biosynthetic Gene Clusters, Heterologous Chassis, and Synthetic Microbial Consortia

Repurposing the yeast peroxisome to compartmentalize a toxic enzyme enables improved (S)-reticuline production

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