Recent Advances in Re-engineering Modular PKS and NRPS Assembly Lines

Beck, Charlotte; Garzón, Jaime Felipe Guerrero; Weber, Tilmann

doi:10.1007/s12257-020-0265-5

Cited by 37 publications

(32 citation statements)

References 82 publications

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“…All type SN RPSs sharing the same subunit 2o riginating from GxpS (NRPS-5) were functional (Figure 4a:N RPS-11, Figure 4b:N RPS-15 --19, Figure 4c:N RPS-20), independent of subunit 1so rigin (Gram-negative/-positive) and/or C domains acceptor site substrate specificity.F or instance, NRPS-18 (BicA subunit 1 + GxpS subunit 2) and -19 (XldS subunit 1 + GxpS subunit 2), not complying with the XUs specificity rules (Figure 4b), produced peptides 15 ( % 40 mg L À1 ,F igure S14) and 16-19 (0.1-5.5 mg L À1 ,F igure S15), respectively.T he latter peptides (16)(17)(18)(19)only differ in the N-terminal acyl starter unit, originating from the E. coli fatty-acid pool, as also observed in the original xenolindicins. [35] Especially NRPS-18 was expected to be inactive,a s previous studies have shown that the BicA C3 domains acceptor site is highly specific for Arg and cannot process Phe or Leu when covalently fused to subunit 2.…”

Section: Forschungsartikelmentioning

confidence: 88%

“…[11,16,17] Although early engineering attempts,i ncluding the exchange of DDs,t he targeted modification of the Ad omains substrate specificity conferring AA residues,a nd the substitution of domains as well as whole modules,g ave mixed results,s everal notable advances have been published recently. [17,18] To give but one example,w ec omprehensively analysed structural data as well as inter-domain linkers in NRPSs to define novel fusion sites and to provide guidelines for exchanging A-T-C units,d enoted as eXchange Units (XUs), [19] as opposed to canonical modules (C-A-T). [20] The XU concept is based on aconserved W]-[NATEmotif within the C-A linker region that can be leveraged as splicing point to functionally recombine type AN RPSs.…”

Section: Introductionmentioning

confidence: 99%

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Synthetic Zippers as an Enabling Tool for Engineering of Non‐Ribosomal Peptide Synthetases**

et al. 2021

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Non-ribosomal peptide synthetases (NRPSs) are the origin of aw ide range of natural products,i ncluding many clinically used drugs.Efficient engineering of these often giant biosynthetic machineries to produce novel non-ribosomal peptides (NRPs) is an ongoing challenge.H ere we describe ac loning and co-expression strategy to functionally combine NRPS fragments of Gram-negative and -positive origin, synthesising novel peptides at titres up to 220 mg L À1 .E xtending from the recently introduced definition of eXchange Units (XUs), we inserted synthetic zippers (SZs) to split single protein NRPSs into independently expressed and translated polypeptide chains.T hese synthetic type of NRPS (type S) enables easier access to engineering,o vercomes cloning limitations,a nd provides as imple and rapid approach to building peptide libraries via the combination of different NRPS subunits.

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Section: Forschungsartikelmentioning

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Synthetic Zippers as an Enabling Tool for Engineering of Non‐Ribosomal Peptide Synthetases**

et al. 2021

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“…Besides AA substitution, the swapping of the functional domains or motifs also serves as an approach to altering the substrate acceptance and product spectrum [63,64]. An NRPS, BbBEAS, controls the chain length of its catalytic product through the function of the C-terminal domain.…”

Section: Re-purposing Biosynthetic Enzymes For Non-native Compound Synthesismentioning

confidence: 99%

Harnessing the yeast Saccharomyces cerevisiae for the production of fungal secondary metabolites

Wang

Kell

Borodina

2021

Essays in Biochemistry

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Fungal secondary metabolites (FSMs) represent a remarkable array of bioactive compounds, with potential applications as pharmaceuticals, nutraceuticals, and agrochemicals. However, these molecules are typically produced only in limited amounts by their native hosts. The native organisms may also be difficult to cultivate and genetically engineer, and some can produce undesirable toxic side-products. Alternatively, recombinant production of fungal bioactives can be engineered into industrial cell factories, such as aspergilli or yeasts, which are well amenable for large-scale manufacturing in submerged fermentations. In this review, we summarize the development of baker’s yeast Saccharomyces cerevisiae to produce compounds derived from filamentous fungi and mushrooms. These compounds mainly include polyketides, terpenoids, and amino acid derivatives. We also describe how native biosynthetic pathways can be combined or expanded to produce novel derivatives and new-to-nature compounds. We describe some new approaches for cell factory engineering, such as genome-scale engineering, biosensor-based high-throughput screening, and machine learning, and how these tools have been applied for S. cerevisiae strain improvement. Finally, we prospect the challenges and solutions in further development of yeast cell factories to more efficiently produce FSMs.

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“…The metabolites that use these conserved machines such as polyketides (PK), which is synthesized by polyketide synthase (PKS), and non-ribosomal synthesized peptide (NRP), produced by non-ribosomal peptide synthetase (NRPS) (Ziemert et al 2016). The wellknown polyketide class, such as the antibiotic erythromycin or the immunosuppressant rapamycin are bio-synthesized by PKS whereas the non-ribosomal peptide class compound, such as penicillin antibiotics is bio-synthesized by NRPS (Beck et al 2020).…”

Section: Introductionmentioning

confidence: 99%

Antiplasmodial activity, biosynthetic gene clusters diversity, and secondary metabolite constituent of selected Indonesian Streptomyces

et al. 2021

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Abstract. Damayanti E, Lisdiyanti P, Sundowo A, Ratnakomala S, Dinoto A, Widada J, Mustofa. 2021. Antiplasmodial activity, biosynthetic gene clusters diversity, and secondary metabolite constituent of selected Indonesian Streptomyces. Biodiversitas 22: 3478-3487. Actinobacteria of the genus Streptomyces are known as the primary candidate antibiotics, but still limited for antiplasmodial drugs. This study aimed to investigate the antiplasmodial activity, the biosynthetic gene clusters (BGCs) diversity, and the secondary metabolites constituent of selected Indonesian Streptomyces. The bacteria were isolated from various habitats: karst soil (GMR22), mangrove sediments (BSE7F and SHP 22-7), and marine sediment (GMY01). Molecular identification by 16S rDNA sequencing were performed for confirmation and morphological characterization by scanning electron microscope (SEM) were performed for identification. In vitro antiplasmodial assay was performed on human Plasmodium falciparum FCR-3. The BGCs which encode secondary metabolites were analysed using antiSMASH version 5 based on available whole genome sequence (WGS) data. The secondary metabolites were obtained from liquid fermentation followed by extraction using methanol and ethyl acetate. The secondary metabolites constituent was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). The molecular identification showed that GMR22 had similarity to Streptomyces lactacystinicus (98.02%), while BSE7F was similar to Streptomyces althioticus (97.06%), SHP 22-7 was similar to Streptomyces rochei (94.84%), and GMY01 to Streptomyces odonnellii (98.57%). All of isolates had morphological characteristics as the genus Streptomyces bacteria. The highest Plasmodium inhibition (81.84 ± 3.5%) was demonstrated by ethyl acetate extract of marine-derived Streptomyces sp. GMY01 (12.5 µg/mL). Non-ribosomal polyketide synthetase (NRPS), polyketide synthase (PKS) and hybrid of NRPS-PKS were the major BGCs in all Streptomyces. Majority of the Streptomyces produced compounds containing CHON elements with molecular weight approximately 100-400 Da. The active extract of GMY01 bacterium had five major detected compounds, namely kuraramine (C12H18N2O2), laminine (C9H20N2O2) 2-ethylacetanilide (C10H13NO), propoxur (C11H15NO3), and 3-methyl-1,2-diphenylbutan-1-one (C17H18O). This Indonesian marine bacterium is potential for bioassay guided isolation of antiplasmodial compounds in the future studies.

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Recent Advances in Re-engineering Modular PKS and NRPS Assembly Lines

Cited by 37 publications

References 82 publications

Synthetic Zippers as an Enabling Tool for Engineering of Non‐Ribosomal Peptide Synthetases**

Synthetic Zippers as an Enabling Tool for Engineering of Non‐Ribosomal Peptide Synthetases**

Harnessing the yeast Saccharomyces cerevisiae for the production of fungal secondary metabolites

Antiplasmodial activity, biosynthetic gene clusters diversity, and secondary metabolite constituent of selected Indonesian Streptomyces

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