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Diphenyl ethers (DPEs) are produced by filamentous fungi using polyketide synthases (PKSs) directly, or via Cu oxidase-catalyzed oxidative rearrangements of benzophenone intermediates. Here, we use heterologous expression to reveal a third route towards DPEs in Preussia isomera that relies on an oxidative multienzyme cascade to convert a PKS-generated, ester-linked didepside to depsidones and further to DPEs, and apply comparative genomics to identify conserved biosynthetic gene clusters for this pathway in multiple fungi. The distribution of DPE products is modulated by the expression chassis upon pathway reconstitution. Among the post-PKS enzymes, the DpeH tyrosinase shows considerable substrate promiscuity towards synthetic DPE analogues. By creating hybrid enzymes with a DpeH orthologue from Aspergillus nidulans, we identify the C-terminal region of DpeH to alter substrate recognition. Our work highlights an evolutionarily conserved way to produce DPEs, and provides enzymatic tools to generate DPE analogues with broad spectrum antibiotic activity against multidrug-resistant human pathogens.
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Unnatural product (uNP) nonribosomal peptides promise to be a valuable source of pharmacophores for drug discovery. However, the extremely large size and complexity of the nonribosomal peptide synthetase (NRPS) enzymes pose formidable challenges to the production of such uNPs by combinatorial biosynthesis and synthetic biology. Here we report a new NRPS dissection strategy that facilitates the engineering and heterologous production of these NRPSs. This strategy divides NRPSs into "splitting units", each forming an enzyme subunit that contains catalytically independent modules. Functional collaboration between the subunits is then facilitated by artificially duplicating, at the N-terminus of the downstream subunit, the linker - thiolation domain - linker fragment that is resident at the C-terminus of the upstream subunit. Using the suggested split site that follows a conserved motif in the linker connecting the adenylation and the thiolation domains allows cognate or chimeric splitting unit pairs to achieve productivities that match, and in many cases surpass those of hybrid chimeric enzymes, and even those of intact NRPSs, upon production in a heterologous chassis. Our strategy provides facile options for the rational engineering of fungal NRPSs and for the combinatorial reprogramming of nonribosomal peptide production.
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Péptido Sintasas , Ingeniería de Proteínas , Péptido Sintasas/metabolismo , Péptido Sintasas/química , Péptido Sintasas/genética , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Some species of the genus Aegilops, a wild relative of wheat, carry chromosomes that after introducing to wheat exhibit preferential transmission to progeny. Their selective retention is a result of the abortion of gametes lacking them due to induced chromosomal aberrations. These chromosomes are termed Gametocidal (Gc) and, based on their effects, they are categorized into three types: mild, intense or severe, and very strong. Gc elements within the same homoeologous chromosome groups of Aegilops (II, III, or IV) demonstrate similar Gc action. This review explores the intriguing dynamics of Gc chromosomes and encompasses comprehensive insights into their source species, behavioral aspects, mode of action, interactions, suppressions, and practical applications of the Gc system in wheat breeding. By delving into these areas, this work aims to contribute to the development of novel plant genetic resources for wheat breeding. The insights provided herein shed light on the utilization of Gc chromosomes to produce chromosomal rearrangements in wheat and its wild relatives, thereby facilitating the generation of chromosome deletions, translocations, and telosomic lines. The Gc approach has significantly advanced various aspects of wheat genetics, including the introgression of novel genes and alleles, molecular markers and gene mapping, and the exploration of homoeologous relationships within Triticeae species. The mystery lies in why gametes possessing Gc genes maintain their normality while those lacking Gc genes suffer abnormalities, highlighting an unresolved research gap necessitating deeper investigation.
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Polyketides with the isochroman-3-one pharmacophore are rare among fungal natural products as their biosynthesis requires an unorthodox S-type aromatic ring cyclization. Genome mining uncovered a conserved gene cluster in select leotiomycetous fungi that encodes the biosynthesis of cytosporones, including isochroman-3-one congeners. Combinatorial biosynthesis in total biosynthetic and biocatalytic formats in Saccharomyces cerevisiae and in vitro reconstitution of key reactions with purified enzymes revealed how cytosporone structural and bioactivity diversity is generated. The S-type acyl dihydroxyphenylacetic acid (ADA) core of cytosporones is assembled by a collaborating polyketide synthase pair. Thioesterase domain-catalyzed transesterification releases ADA esters, some of which are known Nur77 modulators. Alternatively, hydrolytic release allows C6 hydroxylation by a flavin-dependent monooxygenase, yielding a trihydroxybenzene moiety. Reduction of the C9 carbonyl by a short chain dehydrogenase/reductase initiates isochroman-3-one formation, affording cytosporones with cytotoxic and antimicrobial activity. Enoyl di- or trihydroxyphenylacetic acids are generated as shunt products, while isocroman-3,4-diones are formed by autoxidation. The cytosporone pathway offers novel polyketide biosynthetic enzymes for combinatorial synthetic biology to advance the production of "unnatural" natural products for drug discovery.
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Productos Biológicos , Policétidos , Hongos/genética , Saccharomyces cerevisiae/metabolismo , Sintasas Poliquetidas/metabolismo , Policétidos/química , Productos Biológicos/metabolismoRESUMEN
Powdery mildew poses a significant threat to wheat crops worldwide, emphasizing the need for durable disease control strategies. The wheat-Dasypyrum villosum T5AL·5 V#4 S and T5DL·5 V#4 S translocation lines carrying powdery mildew resistant gene Pm55 shows developmental-stage and tissue-specific resistance, whereas T5DL·5 V#5 S line carrying Pm5V confers resistance at all stages. Here, we clone Pm55 and Pm5V, and reveal that they are allelic and renamed as Pm55a and Pm55b, respectively. The two Pm55 alleles encode coiled-coil, nucleotide-binding site-leucine-rich repeat (CNL) proteins, conferring broad-spectrum resistance to powdery mildew. However, they interact differently with a linked inhibitor gene, SuPm55 to cause different resistance to wheat powdery mildew. Notably, Pm55 and SuPm55 encode unrelated CNL proteins, and the inactivation of SuPm55 significantly reduces plant fitness. Combining SuPm55/Pm55a and Pm55b in wheat does not result in allele suppression or yield penalty. Our results provide not only insights into the suppression of resistance in wheat, but also a strategy for breeding durable resistance.
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Ascomicetos , Triticum , Triticum/genética , Alelos , Ascomicetos/genética , Fitomejoramiento , Poaceae/genética , Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/genéticaRESUMEN
Tiller number is a key component of wheat plant architecture having a direct impact on grain yield. Because of their viability, biotic resistance, and abiotic stress tolerance, wild relative species are a valuable gene source for increasing wheat genetic diversity, including yield potential. Agropyron glael, a perennial hybrid of Thinopyrum intermedium and Th. ponticum, was created in the 1930s. Recent genome analyses identified five evolutionarily distinct subgenomes (J, Jst, Jvs, Jr, and St), making A. glael an important gene source for transferring useful agronomical traits into wheat. During a bread wheat × A. glael crossing program, a genetically stable translocation line, WT153397, was developed. Sequential in situ hybridizations (McGISH) with J-, St-, and D-genomic DNA probes and pSc119.2, Afa family, pTa71, and (GAA)7 DNA repeats, as well as molecular markers specific for the wheat 6D chromosome, revealed the presence of a 6DS.6Jvs Robertsonian translocation in the genetic line. Field trials in low-input and high-input breeding nurseries over four growing seasons demonstrated the Agropyron chromosome arm's high compensating ability for the missing 6DL, as spike morphology and fertility of WT153397 did not differ significantly from those of wheat parents, Mv9kr1 and 'Mv Karizma.' Moreover, the introgressed 6Jvs chromosome arm significantly increased the number of productive tillers, resulting in a significantly higher grain yield potential compared to the parental wheat cultivars. The translocated chromosome could be highly purified by flow cytometric sorting due to the intense fluorescent labeling of (GAA)7 clusters on the Thinopyrum chromosome arm, providing an opportunity to use chromosome genomics to identify Agropyron gene variant(s) responsible for the tillering capacity. The translocation line WT153397 is an important genetic stock for functional genetic studies of tiller formation and useful breeding material for increasing wheat yield potential. The study also discusses the use of the translocation line in wheat breeding. Supplementary information: The online version contains supplementary material available at 10.1007/s11032-024-01439-y.
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Plant nucleotide-binding leucine-rich repeat (NLRs) immune receptors directly or indirectly recognize pathogen-secreted effector molecules to initiate plant defense. Recognition of multiple pathogens by a single NLR is rare and usually occurs via monitoring for changes to host proteins; few characterized NLRs have been shown to recognize multiple effectors. The barley (Hordeum vulgare) NLR gene Mildew locus a (Mla) has undergone functional diversification, and the proteins encoded by different Mla alleles recognize host-adapted isolates of barley powdery mildew (Blumeria graminis f. sp. hordei [Bgh]). Here, we show that Mla3 also confers resistance to the rice blast fungus Magnaporthe oryzae in a dosage-dependent manner. Using a forward genetic screen, we discovered that the recognized effector from M. oryzae is Pathogenicity toward Weeping Lovegrass 2 (Pwl2), a host range determinant factor that prevents M. oryzae from infecting weeping lovegrass (Eragrostis curvula). Mla3 has therefore convergently evolved the capacity to recognize effectors from diverse pathogens.
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Ascomicetos , Eragrostis , Hordeum , Magnaporthe , Virulencia/genética , Hordeum/genética , Eragrostis/metabolismo , Plantas/metabolismo , Especificidad del Huésped , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Gene cloning in repeat-rich polyploid genomes remains challenging. Here, we describe a strategy for overcoming major bottlenecks in cloning of the powdery mildew resistance gene (R-gene) Pm69 derived from tetraploid wild emmer wheat. A conventional positional cloning approach was not effective owing to suppressed recombination. Chromosome sorting was compromised by insufficient purity. A Pm69 physical map, constructed by assembling Oxford Nanopore Technology (ONT) long-read genome sequences, revealed a rapidly evolving nucleotide-binding leucine-rich repeat (NLR) R-gene cluster with structural variations. A single candidate NLR was identified by anchoring RNA sequencing reads from susceptible mutants to ONT contigs and was validated by virus-induced gene silencing. Pm69 is likely a newly evolved NLR and was discovered in only one location across the wild emmer wheat distribution range in Israel. Pm69 was successfully introgressed into cultivated wheat, and a diagnostic molecular marker was used to accelerate its deployment and pyramiding with other R-genes.
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Genes de Plantas , Triticum , Triticum/genética , Genes de Plantas/genética , Mapeo Cromosómico , Clonación Molecular , Familia de MultigenesRESUMEN
The annual goatgrass, Aegilops biuncialis is a rich source of genes with considerable agronomic value. This genetic potential can be exploited for wheat improvement through interspecific hybridization to increase stress resistance, grain quality and adaptability. However, the low throughput of cytogenetic selection hampers the development of alien introgressions. Using the sequence of flow-sorted chromosomes of diploid progenitors, the present study enabled the development of chromosome-specific markers. In total, 482 PCR markers were validated on wheat (Mv9kr1) and Ae. biuncialis (MvGB642) crossing partners, and 126 on wheat-Aegilops additions. Thirty-two markers specific for U- or M-chromosomes were used in combination with GISH and FISH for the screening of 44 Mv9kr1 × Ae. biuncialis BC3F3 genotypes. The predominance of chromosomes 4M and 5M, as well as the presence of chromosomal aberrations, may indicate that these chromosomes have a gametocidal effect. A new wheat-Ae. biuncialis disomic 4U addition, 4M(4D) and 5M(5D) substitutions, as well as several introgression lines were selected. Spike morphology and fertility indicated that the Aegilops 4M or 5M compensated well for the loss of 4D and 5D, respectively. The new cytogenetic stocks represent valuable genetic resources for the introgression of key genes alleles into wheat.
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Aegilops , Triticum , Triticum/genética , Aegilops/genética , Hibridación Fluorescente in Situ , Cromosomas de las Plantas/genética , Translocación Genética , Marcadores Genéticos , GenómicaRESUMEN
Saccharomyces cerevisiae has been extensively used as a convenient synthetic biology chassis to reconstitute fungal polyketide biosynthetic pathways. Despite progress in refactoring these pathways for expression and optimization of the yeast production host by metabolic engineering, product yields often remain unsatisfactory. Such problems are especially acute when synthetic biological production is used for bioprospecting via genome mining or when chimeric fungal polyketide synthases (PKSs) are employed to produce novel bioactive compounds. In this work, we demonstrate that empirically balancing the expression levels of the two collaborating PKS subunits that afford benzenediol lactone (BDL)-type fungal polyketides is a facile strategy to improve the product yields. This is accomplished by systematically and independently altering the copy numbers of the two plasmids that express these PKS subunits. We applied this plasmid copy number engineering strategy to two orphan PKSs from genome mining where the yields of the presumed BDL products in S. cerevisiae were far too low for product isolation. This optimization resulted in product yield improvements of up to 10-fold, allowing for the successful isolation and structure elucidation of new BDL analogues. Heterocombinations of these PKS subunits from genome mining with those from previously identified BDL pathways led to the combinatorial biosynthesis of several additional novel BDL-type polyketides.
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Policétidos , Policétidos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Variaciones en el Número de Copia de ADN , Sintasas Poliquetidas/metabolismo , Lactonas/metabolismo , Plásmidos/genéticaRESUMEN
Synthetic biology requires efficient systems that support the well-coordinated co-expression of multiple genes. Here, we discover a 9-bp nucleotide sequence that enables efficient polycistronic gene expression in yeasts and filamentous fungi. Coupling polycistronic expression to multiplexed, markerless, CRISPR/Cas9-based genome editing, we develop a strategy termed HACKing (Highly efficient and Accessible system by CracKing genes into the genome) for the assembly of multigene pathways. HACKing allows the expression level of each enzyme to be precalibrated by linking their translation to those of host proteins with predetermined abundances under the desired fermentation conditions. We validate HACKing by rapidly constructing highly efficient Saccharomyces cerevisiae cell factories that express 13 biosynthetic genes, and produce model endogenous (1,090.41 ± 80.92 mg L-1 squalene) or heterologous (1.04 ± 0.02 mg L-1 mogrol) terpenoid products. Thus, HACKing addresses the need of synthetic biology for predictability, simplicity, scalability, and speed upon fungal pathway engineering for valuable metabolites.
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Sistemas CRISPR-Cas , Edición Génica , Saccharomyces cerevisiae/genética , Hongos/genéticaRESUMEN
Background: Advanced glycation end products (AGEs) are heterogenous group of irreversible chemical moieties originated from non-enzymatic glycation and oxidation of proteins, nucleic acids, and lipids. The engagement of AGEs with their chief cellular receptor (RAGE) activates a myriad of signaling pathways contributing to the progression of chronic diseases like autoimmune thyroiditis, type 2 diabetes mellitus and its complications. Soluble RAGE (sRAGE) prevents AGE-RAGE interaction in a competitive manner. Objective: We investigated the association between serum AGE, sRAGE and thyroid function in 73 Hashimoto thyroiditis patients (HT) on levothyroxine substitution, and in 83 age, BMI and gender-matched healthy controls. Methods: The serum AGEs levels were determined by autofluorescence on a multi-mode microplate reader, and the serum sRAGE levels by ELISA method. Results: Mean AGE level was lower (10.71 vs 11.45 AU/µg protein; p=0.046), while mean sRAGE level was higher (923 vs 755 pg/mL; p<0.0005) in the serum of HT patients than the controls. AGE correlated with age, while sRAGE correlated negatively with BMI in both groups. We found negative correlation between AGE and fT3 levels (r=-0.32; p=0.006) and sRAGE and TSH levels (r=-0.27; p=0.022) in HT patients, while we failed to find association between AGE, sRAGE and parameters of thyroid function in the control group. Median AGE/sRAGE ratio was lower in HT patients than in controls (2.4, IQR 1.9 - 3.1 vs 3.3, IQR 2.3 - 4.1 AU/pg; p < 0.001). In HT patients, the AGE/sRAGE ratio correlated positively with BMI and correlated negatively with fT3. Conclusion: According to our results in HT patients lower TSH and higher fT3 levels within the reference range is accompanied by a favorable AGE/RAGE balance. Further investigations are needed to confirm these results.
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Diabetes Mellitus Tipo 2 , Enfermedad de Hashimoto , Humanos , Enfermedad de Hashimoto/tratamiento farmacológico , Tiroxina , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Productos Finales de Glicación Avanzada , TirotropinaRESUMEN
Root-knot nematodes (RKNs) cause significant economic damage to crop plants, spurring demand for safe, affordable, and sustainable nematicides. A previous study by our research team showed that the combination of two nematicidal secondary metabolites (SMs) derived from Photorhabdus bacteria, trans-cinnamic acid (t-CA), and (4E)-5-phenylpent-4-enoic acid (PPA) have a synergistic effect against RKNs in vitro. In this study, we considered in planta assays to assess the effects of this SM mixture on the virulence and reproductive fitness of the RKN Meloidogyne incognita in a cowpea. Factorial combinations of five t-CA + PPA concentrations (0, 9.0, 22.9, 57.8, and 91.0 µg/ml) and two nematode inoculation conditions (presence or absence) were evaluated in 6-week growth chamber experiments. Results from this study showed that a single root application of the t-CA + PPA mixture significantly reduced the penetration of M. incognita infective juveniles (J2s) into the cowpea roots. The potential toxicity of t-CA + PPA on RKN-susceptible cowpea seedlings was also investigated. The effect of t-CA + PPA × nematode inoculation interactions and the t-CA + PPA mixture did not show significant phytotoxic effects, nor did it adversely affect plant growth parameters or alter leaf chlorophyll content. Total leaf chlorophyll and chlorophyll b content were significantly reduced (by 15 and 22%, respectively) only by the nematode inoculum and not by any of the SM treatments. Our results suggest that a single root application of a mixture of t-CA and PPA reduces M. incognita J2's ability to infect the roots without impairing plant growth or chlorophyll content.
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Photorhabdus , Tylenchoidea , Vigna , Animales , Antinematodos/farmacología , ClorofilaRESUMEN
Flow cytometry offers a unique way of analyzing and manipulating plant chromosomes. During a rapid movement in a liquid stream, large populations can be classified in a short time according to their fluorescence and light scatter properties. Chromosomes whose optical properties differ from other chromosomes in a karyotype can be purified by flow sorting and used in a range of applications in cytogenetics, molecular biology, genomics, and proteomics. As the samples for flow cytometry must be liquid suspensions of single particles, intact chromosomes must be released from mitotic cells. This protocol describes a procedure for preparation of suspensions of mitotic metaphase chromosomes from meristem root tips and their flow cytometric analysis and sorting for various downstream applications.
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Cromosomas de las Plantas , Cromosomas , Citometría de Flujo/métodos , Suspensiones , Citogenética , CariotipificaciónRESUMEN
The introgression of chromosome segments from wild relatives is an established strategy to enrich crop germplasm with disease-resistance genes1. Here we use mutagenesis and transcriptome sequencing to clone the leaf rust resistance gene Lr9, which was introduced into bread wheat from the wild grass species Aegilops umbellulata2. We established that Lr9 encodes an unusual tandem kinase fusion protein. Long-read sequencing of a wheat Lr9 introgression line and the putative Ae. umbellulata Lr9 donor enabled us to assemble the ~28.4-Mb Lr9 translocation and to identify the translocation breakpoint. We likewise cloned Lr58, which was reportedly introgressed from Aegilops triuncialis3, but has an identical coding sequence compared to Lr9. Cytogenetic and haplotype analyses corroborate that the two genes originate from the same translocation event. Our work sheds light on the emerging role of kinase fusion proteins in wheat disease resistance, expanding the repertoire of disease-resistance genes for breeding.
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Basidiomycota , Triticum , Triticum/genética , Genes de Plantas , Fitomejoramiento , Poaceae/genética , Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/genética , Basidiomycota/genéticaRESUMEN
To safeguard bread wheat against pests and diseases, breeders have introduced over 200 resistance genes into its genome, thus nearly doubling the number of designated resistance genes in the wheat gene pool1. Isolating these genes facilitates their fast-tracking in breeding programs and incorporation into polygene stacks for more durable resistance. We cloned the stem rust resistance gene Sr43, which was crossed into bread wheat from the wild grass Thinopyrum elongatum2,3. Sr43 encodes an active protein kinase fused to two domains of unknown function. The gene, which is unique to the Triticeae, appears to have arisen through a gene fusion event 6.7 to 11.6 million years ago. Transgenic expression of Sr43 in wheat conferred high levels of resistance to a wide range of isolates of the pathogen causing stem rust, highlighting the potential value of Sr43 in resistance breeding and engineering.
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Basidiomycota , Resistencia a la Enfermedad , Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/genética , Fitomejoramiento , Genes de Plantas , Basidiomycota/genéticaRESUMEN
Monascus pigments (MPs) display many beneficial biological activities and have been widely utilized as natural food-grade colorants in the food processing industry. The presence of the mycotoxin citrinin (CIT) seriously restricts the application of MPs, but the gene regulation mechanisms governing CIT biosynthesis remain unclear. We performed a RNA-Seq-based comparative transcriptomic analysis of representative high MPs-producing Monascus purpureus strains with extremely high vs. low CIT yields. In addition, we performed qRT-PCR to detect the expression of genes related to CIT biosynthesis, confirming the reliability of the RNA-Seq data. The results revealed that there were 2518 differentially expressed genes (DEGs; 1141 downregulated and 1377 upregulated in the low CIT producer strain). Many upregulated DEGs were associated with energy metabolism and carbohydrate metabolism, with these changes potentially making more biosynthetic precursors available for MPs biosynthesis. Several potentially interesting genes that encode transcription factors were also identified amongst the DEGs. The transcriptomic results also showed that citB, citD, citE, citC and perhaps MpigI were key candidate genes to limit CIT biosynthesis. Our studies provide useful information on metabolic adaptations to MPs and CIT biosynthesis in M. purpureus, and provide targets for the fermentation industry towards the engineering of safer MPs production.
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Bacterial vaginosis (BV) is a common vaginal disease associated with abnormal changes in the vaginal microbiome. Our previous study found that Lactobacillus rhamnosus has a good therapeutic effect on bacterial vaginosis by inhibiting the most prominent bacterium associated with BV, Gardnerella vaginalis. In this study, we show that acetic acid and lactic acid are the main substances in the cell-free supernatant (CFS) of L. rhamnosus that inhibit the growth of G. vaginalis. Further study on the mechanism showed that acetic acid and lactic acid alter the morphology of the G. vaginalis cells, eventually causing the cells to shrink or burst, resulting in exudation of their intracellular contents. In addition, these two organic acids also dissipate the membrane potential of bacterial cells, affecting their synthesis of ATP. A reduced activity of the Na+/K+-ATPase leads to abnormal ATP metabolism, and ultimately inhibits the growth and reproduction of G. vaginalis. Our study provides valuable information for the widespread application of L. rhamnosus in the treatment of bacterial vaginosis.
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Antiinfecciosos , Lacticaseibacillus rhamnosus , Vaginosis Bacteriana , Humanos , Femenino , Gardnerella vaginalis , Vaginosis Bacteriana/tratamiento farmacológico , Vaginosis Bacteriana/microbiología , Vagina/microbiología , Ácido Acético , Adenosina TrifosfatoRESUMEN
Orsellinic acid (OA) derivatives are produced by filamentous fungi using nonreducing polyketide synthases (nrPKSs). The chain-releasing thioesterase (TE) domains of such nrPKSs were proposed to also catalyze dimerization to yield didepsides, such as lecanoric acid. Here, we use combinatorial domain exchanges, domain dissections and reconstitutions to reveal that the TE domain of the lecanoric acid synthase Preu6 of Preussia isomera must collaborate with the starter acyl transferase (SAT) domain from the same nrPKS. We show that artificial SAT-TE fusion proteins are highly effective catalysts and reprogram the ketide homologation chassis to form didepsides. We also demonstrate that dissected SAT and TE domains of Preu6 physically interact, and SAT and TE domains of OA-synthesizing nrPKSs may co-evolve. Our work highlights an unexpected domain-domain interaction in nrPKSs that must be considered for the combinatorial biosynthesis of unnatural didepsides, depsidones, and diphenyl ethers.
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Ascomicetos , Sintasas Poliquetidas , Sintasas Poliquetidas/metabolismo , Aciltransferasas , Ascomicetos/metabolismoRESUMEN
Covering: 2011 up to the end of 2021.Fungal nonribosomal peptides (NRPs) and the related polyketide-nonribosomal peptide hybrid products (PK-NRPs) are a prolific source of bioactive compounds, some of which have been developed into essential drugs. The synthesis of these complex natural products (NPs) utilizes nonribosomal peptide synthetases (NRPSs), multidomain megaenzymes that assemble specific peptide products by sequential condensation of amino acids and amino acid-like substances, independent of the ribosome. NRPSs, collaborating polyketide synthase modules, and their associated tailoring enzymes involved in product maturation represent promising targets for NP structure diversification and the generation of small molecule unnatural products (uNPs) with improved or novel bioactivities. Indeed, reprogramming of NRPSs and recruiting of novel tailoring enzymes is the strategy by which nature evolves NRP products. The recent years have witnessed a rapid development in the discovery and identification of novel NRPs and PK-NRPs, and significant advances have also been made towards the engineering of fungal NRP assembly lines to generate uNP peptides. However, the intrinsic complexities of fungal NRP and PK-NRP biosynthesis, and the large size of the NRPSs still present formidable conceptual and technical challenges for the rational and efficient reprogramming of these pathways. This review examines key examples for the successful (and for some less-successful) re-engineering of fungal NRPS assembly lines to inform future efforts towards generating novel, biologically active peptides and PK-NRPs.