Metabolic engineering of the shikimate pathway in Amycolatopsis strains for optimized glycopeptide antibiotic production.

Glycopeptide antibiotics (GPA) consist of a glycosylated heptapeptide backbone enriched in aromatic residues originating from the shikimate pathway. Since the enzymatic reactions within the shikimate pathway are highly feedback-regulated, this raises the question as to how GPA producers control the delivery of precursors for GPA assembly. We chose Amycolatopsis balhimycina, the producer of balhimycin, as a model strain for analyzing the key enzymes of the shikimate pathway. A. balhimycina contains two copies each of the key enzymes of the shikimate pathway, deoxy-d-arabino-heptulosonate-7-phosphate synthase (Dahp) and prephenate dehydrogenase (Pdh), with one pair (Dahpsec and Pdhsec) encoded within the balhimycin biosynthetic gene cluster and one pair (Dahpprim and Pdhprim) in the core genome. While overexpression of the dahpsec gene resulted in a significant (>4-fold) increase in balhimycin yield, no positive effects were observed after overexpression of the pdhprim or pdhsec genes. Investigation of allosteric enzyme inhibition revealed that cross-regulation between the tyrosine and phenylalanine pathways plays an important role. Tyrosine, a key precursor of GPAs, was found to be a putative activator of prephenate dehydratase (Pdt), which catalyzes the first step reaction from prephenate to phenylalanine in the shikimate pathway. Surprisingly, overexpression of pdt in A. balhimycina led to an increase in antibiotic production in this modified strain. In order to demonstrate that this metabolic engineering approach is generally applicable to GPA producers, we subsequently applied this strategy to Amycolatopsis japonicum and improved the production of ristomycin A, which is used in diagnosis of genetic disorders. Comparison of "cluster-specific" enzymes with the isoenzymes from the primary metabolism's pathway provided insights into the adaptive mechanisms used by producers to ensure adequate precursor supply and GPA yields. These insights further demonstrate the importance of a holistic approach in bioengineering efforts that takes into account not only peptide assembly but also adequate precursor supply.

Optimization of FK-506 production in Streptomyces tsukubaensis by modulation of Crp-mediated regulation.

FK-506 is a potent immunosuppressive macrocyclic polyketide with growing pharmaceutical interest, produced by Streptomyces tsukubaensis. However, due to low levels synthesized by the wild-type strain, biotechnological production of FK-506 is rather limited. Optimization strategies to enhance the productivity of S. tsukubaensis by means of genetic engineering have been established. In this work primarily global regulatory aspects with respect to the FK-506 biosynthesis have been investigated with the focus on the global Crp (cAMP receptor protein) regulator. In expression analyses and protein-DNA interaction studies, the role of Crp during FK-506 biosynthesis was elucidated. Overexpression of Crp resulted in two-fold enhancement of FK-506 production in S. tsukubaensis under laboratory conditions. Further optimizations using fermentors proved that the strategy described in this study can be transferred to industrial scale, presenting a new approach for biotechnological FK-506 production. KEY POINTS: • The role of the global Crp (cAMP receptor protein) regulator for FK-506 biosynthesis in S. tsukubaensis was demonstrated • Crp overexpression in S. tsukubaensis was applied as an optimization strategy to enhance FK-506 and FK-520 production resulting in two-fold yield increase.

The Endophytic Fungus Cyanodermella asteris Influences Growth of the Nonnatural Host Plant Arabidopsis thaliana.

Cyanodermella asteris is a fungal endophyte from Aster tataricus, a perennial plant from the northern part of Asia. Here, we demonstrated an interaction of C. asteris with Arabidopsis thaliana, Chinese cabbage, rapeseed, tomato, maize, or sunflower resulting in different phenotypes such as shorter main roots, massive lateral root growth, higher leaf and root biomass, and increased anthocyanin levels. In a variety of cocultivation assays, it was shown that these altered phenotypes are caused by fungal CO2, volatile organic compounds, and soluble compounds, notably astins. Astins A, C, and G induced plant growth when they were individually included in the medium. In return, A. thaliana stimulates the fungal astin C production during cocultivation. Taken together, our results indicate a bilateral interaction between the fungus and the plant. A stress response in plants is induced by fungal metabolites while plant stress hormones induced astin C production of the fungus. Interestingly, our results not only show unidirectional influence of the fungus on the plant but also vice versa. The plant is able to influence growth and secondary metabolite production in the endophyte, even when both organisms do not live in close contact, suggesting the involvement of volatile compounds.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Mechanism-Based Design of the First GlnA4-Specific Inhibitors.

γ-Glutamylamine synthetases are an important class of enzymes that play a key role in glutamate-based metabolism. Methionine sulfoximine (MSO) is a well-established inhibitor for the archetypal glutamine synthetase (GS) but inhibitors for most GS-like enzymes are unknown. Assuming a conserved catalytic mechanism for GS and GS-like enzymes, we explored if subtype-selective inhibitors can be obtained by merging MSO with the cognate substrates of the respective GS-like enzymes. Using GlnA4Sc from Streptomyces coelicolor, an enzyme recently shown to produce γ-glutamylethanolamine, we demonstrate that MSO can be reengineered in a straightforward fashion into potent and selective GlnA4Sc inhibitors. Linkage chemistry as well as linker length between the MSO moiety and the terminal hydroxyl group derived from ethanolamine were in agreement with the postulated phosphorylated catalytic intermediate. The best GlnA4 inhibitor 7 b potently blocked S. coelicolor growth in the presence of ethanolamine as the sole nitrogen source. Our results provide the first GlnA4Sc -specific inhibitors and suggest a general strategy to develop mechanism-based inhibitors for GS-like enzymes.

Antitumor astins originate from the fungal endophyte Cyanodermella asteris living within the medicinal plant Aster tataricus.

Medicinal plants are a prolific source of natural products with remarkable chemical and biological properties, many of which have considerable remedial benefits. Numerous medicinal plants are suffering from wildcrafting, and thus biotechnological production processes of their natural products are urgently needed. The plant Aster tataricus is widely used in traditional Chinese medicine and contains unique active ingredients named astins. These are macrocyclic peptides showing promising antitumor activities and usually containing the highly unusual moiety 3,4-dichloroproline. The biosynthetic origins of astins are unknown despite being studied for decades. Here we show that astins are produced by the recently discovered fungal endophyte Cyanodermella asteris. We were able to produce astins in reasonable and reproducible amounts using axenic cultures of the endophyte. We identified the biosynthetic gene cluster responsible for astin biosynthesis in the genome of C. asteris and propose a production pathway that is based on a nonribosomal peptide synthetase. Striking differences in the production profiles of endophyte and host plant imply a symbiotic cross-species biosynthesis pathway for astin C derivatives, in which plant enzymes or plant signals are required to trigger the synthesis of plant-exclusive variants such as astin A. Our findings lay the foundation for the sustainable biotechnological production of astins independent from aster plants.

A Second Gamma-Glutamylpolyamine Synthetase, GlnA2, Is Involved in Polyamine Catabolism in Streptomyces coelicolor.

Streptomyces coelicolor is a soil bacterium living in a habitat with very changeable nutrient availability. This organism possesses a complex nitrogen metabolism and is able to utilize the polyamines putrescine, cadaverine, spermidine, and spermine and the monoamine ethanolamine. We demonstrated that GlnA2 (SCO2241) facilitates S. coelicolor to survive under high toxic polyamine concentrations. GlnA2 is a gamma-glutamylpolyamine synthetase, an enzyme catalyzing the first step in polyamine catabolism. The role of GlnA2 was confirmed in phenotypical studies with a glnA2 deletion mutant as well as in transcriptional and biochemical analyses. Among all GS-like enzymes in S. coelicolor, GlnA2 possesses the highest specificity towards short-chain polyamines (putrescine and cadaverine), while its functional homolog GlnA3 (SCO6962) prefers long-chain polyamines (spermidine and spermine) and GlnA4 (SCO1613) accepts only monoamines. The genome-wide RNAseq analysis in the presence of the polyamines putrescine, cadaverine, spermidine, or spermine revealed indication of the occurrence of different routes for polyamine catabolism in S. coelicolor involving GlnA2 and GlnA3. Furthermore, GlnA2 and GlnA3 are differently regulated. From our results, we can propose a complemented model of polyamine catabolism in S. coelicolor, which involves the gamma-glutamylation pathway as well as other alternative utilization pathways.

Mining Indonesian Microbial Biodiversity for Novel Natural Compounds by a Combined Genome Mining and Molecular Networking Approach.

Indonesia is one of the most biodiverse countries in the world and a promising resource for novel natural compound producers. Actinomycetes produce about two thirds of all clinically used antibiotics. Thus, exploiting Indonesia's microbial diversity for actinomycetes may lead to the discovery of novel antibiotics. A total of 422 actinomycete strains were isolated from three different unique areas in Indonesia and tested for their antimicrobial activity. Nine potent bioactive strains were prioritized for further drug screening approaches. The nine strains were cultivated in different solid and liquid media, and a combination of genome mining analysis and mass spectrometry (MS)-based molecular networking was employed to identify potential novel compounds. By correlating secondary metabolite gene cluster data with MS-based molecular networking results, we identified several gene cluster-encoded biosynthetic products from the nine strains, including naphthyridinomycin, amicetin, echinomycin, tirandamycin, antimycin, and desferrioxamine B. Moreover, 16 putative ion clusters and numerous gene clusters were detected that could not be associated with any known compound, indicating that the strains can produce novel secondary metabolites. Our results demonstrate that sampling of actinomycetes from unique and biodiversity-rich habitats, such as Indonesia, along with a combination of gene cluster networking and molecular networking approaches, accelerates natural product identification.

A Regulator Based "Semi-Targeted" Approach to Activate Silent Biosynthetic Gene Clusters.

By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel "semi-targeted" approach focusing on activating "silent" BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10.

Engineering of Streptoalloteichus tenebrarius 2444 for Sustainable Production of Tobramycin.

Tobramycin is a broad-spectrum aminoglycoside antibiotic agent. The compound is obtained from the base-catalyzed hydrolysis of carbamoyltobramycin (CTB), which is naturally produced by the actinomycete Streptoalloteichus tenebrarius. However, the strain uses the same precursors to synthesize several structurally related aminoglycosides. Consequently, the production yields of tobramycin are low, and the compound's purification is very challenging, costly, and time-consuming. In this study, the production of the main undesired product, apramycin, in the industrial isolate Streptoalloteichus tenebrarius 2444 was decreased by applying the fermentation media M10 and M11, which contained high concentrations of starch and dextrin. Furthermore, the strain was genetically engineered by the inactivation of the aprK gene (∆aprK), resulting in the abolishment of apramycin biosynthesis. In the next step of strain development, an additional copy of the tobramycin biosynthetic gene cluster (BGC) was introduced into the ∆aprK mutant. Fermentation by the engineered strain (∆aprK_1-17L) in M11 medium resulted in a 3- to 4-fold higher production than fermentation by the precursor strain (∆aprK). The phenotypic stability of the mutant without selection pressure was validated. The use of the engineered S. tenebrarius 2444 facilitates a step-saving, efficient, and, thus, more sustainable production of the valuable compound tobramycin on an industrial scale.

Genetic Engineering in Combination with Semi-Synthesis Leads to a New Route for Gram-Scale Production of the Immunosuppressive Natural Product Brasilicardin†A.

Brasilicardin A (1) consists of an unusual anti/syn/anti-perhydrophenanthrene skeleton with a carbohydrate side chain and an amino acid moiety. It exhibits potent immunosuppressive activity, yet its mode of action differs from standard drugs that are currently in use. Further pre-clinical evaluation of this promising, biologically active natural product is hampered by restricted access to the ready material, as its synthesis requires both a low-yielding fermentation process using a pathogenic organism and an elaborate, multi-step total synthesis. Our semi-synthetic approach included a) the heterologous expression of the brasilicardin A gene cluster in different non-pathogenic bacterial strains producing brasilicardin A aglycone (5) in excellent yield and b) the chemical transformation of the aglycone 5 into the trifluoroacetic acid salt of brasilicardin A (1 a) via a short and straightforward five-steps synthetic route. Additionally, we report the first preclinical data for brasilicardin A.

Metabolic engineering of Amycolatopsis japonicum for optimized production of [S,S]-EDDS, a biodegradable chelator.

The actinomycete Amycolatopsis japonicum is the producer of the chelating compound [S,S]-ethylenediamine-disuccinc acid (EDDS). [S,S]-EDDS is an isomer of ethylenediamine-tetraacetic acid (EDTA), an economically important chelating compound that suffers from an extremely poor degradability. Frequent use of the persistent EDTA in various industrial and domestic applications has caused an accumulation of EDTA in soil as well as in aqueous environments. As a consequence, EDTA is the highest concentrated anthropogenic compound present in water reservoirs. The [S,S]-form of EDDS has chelating properties similar to EDTA, however, in contrast to EDTA it is readily biodegradable. In order to compete with the cost-effective chemical synthesis of EDTA, we aimed to optimize the biotechnological production of [S,S]-EDDS in A. japonicum by using metabolic engineering approaches. Firstly, we integrated several copies of the [S,S]-EDDS biosynthetic genes into the chromosome of A. japonicum and replaced the native zinc responsive promoter with the strong synthetic constitutive promoter SP44*. Secondly, we increased the supply of O-phospho-serine, the direct precursor of [S,S]-EDDS. The combination of these approaches together with the optimized fermentation process led to a significant improvement in [S,S]-EDDS up to 9.8 g/L with a production rate of 4.3 mg/h/g DCW.

Patscanui: an intuitive web interface for searching patterns in DNA and protein data.

Patterns in biological sequences frequently signify interesting features in the underlying molecule. Many tools exist to search for well-known patterns. Less support is available for exploratory analysis, where no well-defined patterns are known yet. PatScanUI (https://patscan.secondarymetabolites.org/) provides a highly interactive web interface to the powerful generic pattern search tool PatScan. The complex PatScan-patterns are created in a drag-and-drop aware interface allowing researchers to do rapid prototyping of the often complicated patterns useful to identifying features of interest.

Maßgeschneiderte Polyketidsynthasen zur Herstellung von Polyketid-Derivaten.

Polyketides (PKs) are secondary metabolites with valuable properties, including antibiotic, antifungal, antiviral, anticancer, and immunosuppressive activity. The biosynthesis of PKs is accomplished by multifunctional megaenzymes, the polyketide synthases (PKSs). The molecular architecture of those remarkable assembly lines provides opportunities for their engineering and generation of PK derivatives with potentially improved pharmacokinetics and/or expanded spectrum of bioactivity.

Diversity of peptidoglycan structure-Modifications and their physiological role in resistance in antibiotic producers.

Peptidoglycan (PG) is a bacteria specific cell surface layer that ensures the bacterial shape and integrity. The two actinomycetes Amycolatopsis balhimycina and Microbispora sp. PTA-5024 are producers of PG targeting antibiotics. To prevent the binding of their secreted product to their own PG, they developed specific self-resistance mechanisms. Modifications of PG, which are applied by both strains, are the introduction of amide-residues at the PG precursors and the alternative crosslinks within the nascent PG. The PG modifications found in Microbispora sp. PTA-5024 seemed to be an intrinsic characteristic of the genus Microbispora, rather than a specific mechanism of NAI-107 resistance. In contrast, the modifications in A. balhimycina represent an alternative way to avoid suicide specific for glycopeptide producers. The different PG modifications reflect the fact that antibiotic producing organisms contain not only one but multiple mechanisms to ensure protection against biologically active molecules produced by themselves.

Regulation of teicoplanin biosynthesis: refining the roles of tei cluster-situated regulatory genes.

Teicoplanin is a frontline glycopeptide antibiotic produced by Actinoplanes teichomyceticus. It is used to treat complicated cases of infection, including pediatric ones, caused by Gram-positive pathogens. There is a steady interest in elucidating the genetic mechanisms determining teicoplanin production, as they would help overproduce known teicoplanins and discover novel glycopeptides. Herein, we investigate the transcriptional organization of the tei biosynthetic gene cluster and the roles of the cluster-situated regulatory genes in controlling teicoplanin production and self-resistance in A. teichomyceticus. We demonstrate that the tei cluster is organized into nine polygenic and nine monogenic transcriptional units. Most of tei biosynthetic genes are subjected to StrR-like Tei15* control, which, in turn, appears to be regulated by LuxR-type Tei16*. Expression of the genes conferring teicoplanin self-resistance in A. teichomyceticus is not co-regulated with antibiotic production. The gene tei31*, coding for a putative DNA binding protein, is not expressed under teicoplanin producing conditions and is dispensable for antibiotic production. Finally, phylogenesis reconstruction of the glycopeptide cluster-encoded regulators reveals two main clades of StrR-like regulators. Tei15* and close orthologues form one of these clades; the second clade is composed by orthologues of Bbr and Dbv4, governing the biosynthesis of balhimycin and teicoplanin-like A40926, respectively. In addition, the LuxR-type Tei16* appears unrelated to the LuxR-like Dbv3, which is controlling A40926 biosynthesis. Our results shed new light on teicoplanin biosynthesis regulation and on the evolution of novel and old glycopeptide biosynthetic gene clusters.

Comparative statistical component analysis of transgenic, cyanophycin-producing potatoes in greenhouse and field trials.

Potatoes are a promising system for industrial production of the biopolymer cyanophycin as a second compound in addition to starch. To assess the efficiency in the field, we analysed the stability of the system, specifically its sensitivity to environmental factors. Field and greenhouse trials with transgenic potatoes (two independent events) were carried out for three years. The influence of environmental factors was measured and target compounds in the transgenic plants (cyanophycin, amino acids) were analysed for differences to control plants. Furthermore, non-target parameters (starch content, number, weight and size of tubers) were analysed for equivalence with control plants. The huge amount of data received was handled using modern statistical approaches to model the correlation between influencing environmental factors (year of cultivation, nitrogen fertilization, origin of plants, greenhouse or field cultivation) and key components (starch, amino acids, cyanophycin) and agronomic characteristics. General linear models were used for modelling, and standard effect sizes were applied to compare conventional and genetically modified plants. Altogether, the field trials prove that significant cyanophycin production is possible without reduction of starch content. Non-target compound composition seems to be equivalent under varying environmental conditions. Additionally, a quick test to measure cyanophycin content gives similar results compared to the extensive enzymatic test. This work facilitates the commercial cultivation of cyanophycin potatoes.

antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters.

Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.

Elucidation of the zinc-dependent regulation in Amycolatopsis japonicum enabled the identification of the ethylenediamine-disuccinate ([S,S]-EDDS) genes.

The actinomycete Amycolatopsis japonicum produces the complexing agent ethylenediamine-disuccinate ([S,S]-EDDS), which is an isomer of the widely industrially applied ethylenediamine-tetraacetate (EDTA). In contrast to EDTA, [S,S]-EDDS is readily biodegradable and is therefore an alternative with a favourable environmental profile. Biotechnological production of [S,S]-EDDS, however, is not currently possible because its biosynthesis is inhibited by low-micromolar zinc concentrations. Here we illustrate the development of a new strategy for identifying a biosynthetic pathway that is based on the elucidation of transcriptional regulation and the screening for binding sites of the respective regulator that controls the [S,S]-EDDS biosynthesis genes. To achieve this, we identified the zinc uptake regulator Zur in A. japonicum and showed that it mediates the repression of the zinc uptake system ZnuABCAj . The Zur-binding motif, recognized by the zinc-bound Zur protein in the upstream region of znuABCAj , was used to screen the genome, leading to the identification of the aes genes. Transcriptional analysis and shift assays reveal specific zinc-responsive regulation of the aes genes by Zur, and gene inactivation shows their involvement in [S,S]-EDDS biosynthesis. Zur-mediated zinc repression of the [S,S]-EDDS biosynthesis genes is abolished in a Δzur mutant, which offers now the opportunity to develop a biotechnological process.

Distinct mechanisms contribute to immunity in the lantibiotic NAI-107 producer strain Microbispora ATCC PTA-5024.

The investigation of self-resistance in antibiotic producers is important to understand the emergence of antibiotic resistance in pathogens and to improve antibiotic production. Lantibiotics are ribosomally synthesized antibiotics that mostly target peptidoglycan biosynthesis. The actinomycete Microbispora ATCC PTA-5024 produces the lantibiotic NAI-107, which interferes with peptidoglycan biosynthesis by binding bactoprenol-pyrophosphate-coupled peptidoglycan precursors. In order to understand how Microbispora counteracts the action of its own antibiotic, its peptidoglycan composition was analysed in detail. Microbispora peptidoglycan consists of muropeptides with D-Ala and Gly in similar proportion at the fourth position of the peptide stems and alternative 3-3 cross-links besides the classical 4-3 cross-links. In addition, the NAI-107 biosynthetic gene cluster (mlb) was analysed for the expression of immunity proteins. We show that distinct immunity determinants are encoded in the mlb cluster: the ABC transporter MlbYZ acting cooperatively with the transmembrane protein MlbJ and the lipoprotein MlbQ. NMR structural analysis of MlbQ revealed a hydrophobic surface patch, which is proposed to bind the cognate lantibiotic. This study demonstrates that immunity in Microbispora is not only based on one determinant but on the action of the distinct immunity proteins MlbQ, MlbYZ and MlbJ.

The cyclochlorotine mycotoxin is produced by the nonribosomal peptide synthetase CctN in Talaromyces islandicus ('Penicillium islandicum').

Talaromyces islandicus ('Penicillium islandicum') is a widespread foodborne mold that produces numerous secondary metabolites, among them potent mycotoxins belonging to different chemical classes. A notable metabolite is the hepatotoxic and carcinogenic pentapeptide cyclochlorotine that contains the unusual amino acids ß-phenylalanine, 2-aminobutyrate and 3,4-dichloroproline. Although the chemical structure has been known for over five decades, nothing is known about the biosynthetic pathway of cyclochlorotine. Bioinformatic analysis of the recently sequenced genome of T. islandicus identified a wealth of gene clusters potentially coding for the synthesis of secondary metabolites. Here, we show by RNA interference-mediated gene silencing that a nonribosomal peptide synthetase, CctN, is responsible for the synthesis of cyclochlorotine. Moreover, we identified novel cyclochlorotine chemical variants, whose production also depended on cctN expression. Surprisingly, the halogenase required for cyclochlorotine biosynthesis is not encoded in the cct cluster. Nonetheless, our findings enabled us to propose a detailed model for cyclochlorotine biosynthesis. In addition, comparative genomics revealed that cct-like clusters are present in all of the sequenced Talaromyces strains indicating a high prevalence of cyclochlorotine production ability.