Trichoderma harzianum Peptaibols Stimulate Plant Plasma Membrane H+-ATPase Activity.

Because of their ability to promote growth, act as biopesticides, and improve abiotic stress tolerance, Trichoderma spp. have been used for plant seed coating. However, the mechanism for the promotion of plant growth remains unknown. In this study, we investigate the effect of fungal extracts on the plant plasma membrane (PM) H+-ATPase, which is essential for plant growth and often a target of plant-associated microbes. We show that Trichoderma harzianum extract increases H+-ATPase activity, and by fractionation and high-resolution mass spectrometry (MS), we identify the activating components trichorzin PA (tPA) II and tPA VI that belong to the class of peptaibols. Peptaibols are nonribosomal peptides that can integrate into membranes and form indiscriminate ion channels, which causes pesticidal activity. To further investigate peptaibol-mediated H+-ATPase activation, we compare the effect of tPA II and VI to that of the model peptaibol alamethicin (AlaM). We show that AlaM increases H+-ATPase turnover rates in a concentration-dependent manner, with a peak in activity measured at 31.25 µM, above which activity decreases. Using fluorescent probes and light scattering, we find that the AlaM-mediated increase in activity is not correlated to increased membrane fluidity or vesicle integrity, whereas the activity decrease at high AlaM concentrations is likely due to PM overloading of AlaM pores. Overall, our results suggest that the symbiosis of fungi and plants, specifically related to peptaibols, is a concentration-dependent balance, where peptaibols do not act only as biocontrol agents but also as plant growth stimulants.

Solonamides, a Group of Cyclodepsipeptides, Influence Motility in the Native Producer Photobacterium galatheae S2753.

The marine bacterium Photobacterium galatheae S2753 produces a group of cyclodepsipeptides, called solonamides, which impede the virulence but not the survival of Staphylococcus aureus. In addition to their invaluable antivirulence activity, little is known about the biosynthesis and physiological function of solonamides in the native producer. This study generated a solonamide-deficient (Δsol) mutant by in-frame deletion of the sol gene, thereby identifying the core gene for solonamide biosynthesis. By annotation from antiSMASH, the biosynthetic pathway of solonamides in S2753 was also proposed. Mass spectrometry analysis of cell extracts found that deficiency of solonamide production influenced the production of a group of unknown compounds but otherwise did not alter the overall secondary metabolite profile. Physiological comparison between Δsol and wild-type S2753 demonstrated that growth dynamics and biofilm formation of both strains were similar; however, the Δsol mutant displayed reduced motility rings compared to the wild type. Reintroduction of sol restored solonamide production and motility to the mutant, indicating that solonamides influence the motility behavior of P. galatheae S2753. Proteomic analysis of the Δsol and wild-type strains found that eliminating solonamides influenced many cellular processes, including swimming-related proteins and proteins adjusting the cellular cyclic di-GMP concentration. In conclusion, our results revealed the biosynthetic pathway of solonamides and their ecological benefits to P. galatheae S2753 by enhancing motility, likely by altering the motile physiology. IMPORTANCE The broad range of bioactive potentials of cyclodepsipeptides makes these compounds invaluable in the pharmaceutical industry. Recently, a few novel cyclodepsipeptides have been discovered in marine Proteobacteria; however, their biosynthetic pathways remain to be revealed. Here, we demonstrated the biosynthetic genetic basis and pathway of the antivirulence compounds known as solonamides in P. galatheae S2753. This can pave the way for the biological overproduction of solonamides on an industrial scale. Moreover, the comparison of a solonamide-deficient mutant and wild-type S2753 demonstrated that solonamides stimulate the swimming behavior of S2753 and also influence a few key physiological processes of the native producers. These results evidenced that, in addition to their importance as novel drug candidates, these compounds play a pivotal role in the physiology of the producing microorganisms and potentially provide the native producer competitive benefits for their survival in nature.

Phoenicin Switch: Discovering the Trigger for Radical Phoenicin Production in Multiple Wild-Type Penicillium Species.

Society faces the challenge of storing energy from sustainable sources in inexpensive, nontoxic ways that do not deplete the limited resources of Earth. In this regard, quinone redox flow batteries have been proposed as ideal; however, industrially used quinones have traditionally been synthesized from fossil fuels. Therefore, we investigated the production of phoenicin (compound 1), a deep violet dibenzoquinone produced by certain Penicillium species, for its industrial potential. Strains grew as surface cultures on customized growth media with varying production parameters, and phoenicin production was assessed by ultrahigh-performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOF MS) analysis of the supernatant. Phoenicin production was reliant on the sucrose concentration, and by varying that, we produced 4.94 ± 0.56 g/L phoenicin on a Czapek yeast autolysate broth (CY)-based medium with Penicillium phoeniceum (CBS 249.32) as the production host, with 71.91% phoenicin purity in the resulting medium broth. Unexpectedly, metabolites corresponding to phoenicin polymers were tentatively identified in P. phoeniceum, of which the dimer (diphoenicin) was a major chromatographic peak. An MS-based metabolomics study was conducted on P. atrosanguineum using feature-based molecular networking and multivariate statistics, and it was found that few or no known secondary metabolites besides phoenicin were secreted into the growth medium. Finally, the effects of sucrose, sodium nitrate, and yeast extract (YE) in the growth medium were investigated in a 23 full factorial design. The results indicated an optimal sucrose concentration of 92.87 g/L on CY when NaNO3 and YE were fixed at 3 and 5 g/L, respectively. IMPORTANCE This work was undertaken to explore the production of fungal quinones in wild-type strains for use as electrolytes in redox flow batteries. As society converts energy production in a more sustainable direction, it becomes increasingly more important to store sustainable energy in smart ways. Conventional battery technologies imply the use of highly toxic, expensive, and rare metals; thus, quinone redox flow batteries have been proposed to be a desirable alternative. In this study, we explored the possibility of producing the fungal quinone phoenicin in Penicillium spp. by changing the growth parameters. The production of other secondary metabolites and known mycotoxins was also investigated in a metabolomics study. It was shown that phoenicin production was activated by optimizing the carbon concentration of the medium, resulting in high titers and purity of the single metabolite.

Identification and Verification of the Prodigiosin Biosynthetic Gene Cluster (BGC) in Pseudoalteromonas rubra S4059.

Pseudoalteromonas rubra S4059 produces the red pigment prodigiosin, which has pharmaceutical and industrial potential. Here, we targeted a putative prodigiosin-synthesizing transferase PigC, and a pigC in-frame deletion mutant did not produce prodigiosin. However, extractions of the pigC mutant cultures retained antibacterial activity, and bioassay-guided fractionation found antibacterial activity in two fractions of blue color. A precursor of prodigiosin, 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC), was the dominant compound in both the fractions and likely caused the antibacterial activity. Also, a stable blue pigment, di-pyrrolyl-dipyrromethene prodigiosin, was identified from the two fractions. We also discovered antibacterial activity in the sterile filtered (nonextracted) culture supernatant of both wild type and mutant, and both contained a heat-sensitive compound between 30 and 100 kDa. Deletion of prodigiosin production did not affect growth rate or biofilm formation of P. rubra and did not change its fitness, as the mutant and wild type coexisted in equal levels in mixed cultures. In conclusion, a prodigiosin biosynthetic gene cluster (BGC) was identified and verified genetically and chemically in P. rubra S4059 and a stable blue pigment was isolated from the pigC mutant of S4059, suggesting that this strain may produce several prodigiosin-derived compounds of pharmaceutical and/or industrial potential. IMPORTANCE Pigmented Pseudoalteromonas strains are renowned for their production of secondary metabolites, and genome mining has revealed a high number of biosynthetic gene clusters (BGCs) for which the chemistry is unknown. Identification of those BGCs is a prerequisite for linking products to gene clusters and for further exploitation through heterologous expression. In this study, we identified the BGCs for the red, bioactive pigment prodigiosin using genomic, genetic, and metabolomic approaches. We also report here for the first time the production of a stable blue pigment, di-pyrrolyl-dipyrromethene prodigiosin (Dip-PDG), being produced by the pigC mutant of Pseudoalteromonas rubra S4059.

Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium Pseudoalteromonas rubra S4059.

Genome mining of pigmented Pseudoalteromonas has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, Pseudoalteromonas rubra strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented P. rubra S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented Pseudoalteromonas, and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented Pseudoalteromonas, we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential.

Enhancement of antibiotic production by co-cultivation of two antibiotic producing marine Vibrionaceae strains.

Deciphering the cues that stimulate microorganisms to produce their full secondary metabolic potential promises to speed up the discovery of novel drugs. Ecology-relevant conditions, including carbon-source(s) and microbial interactions, are important effectors of secondary metabolite production. Vice versa secondary metabolites are important mediators in microbial interactions, although their exact natural functions are not always completely understood. In this study, we investigated the effects of microbial interactions and in-culture produced antibiotics on the production of secondary metabolites by Vibrio coralliilyticus and Photobacterium galatheae, two co-occurring marine Vibrionaceae. In co-culture, production of andrimid by V. coralliilyticus and holomycin by P. galatheae, were, compared to monocultures, increased 4.3 and 2.7 fold, respectively. Co-cultures with the antibiotic deficient mutant strains (andrimid- and holomycin-) did not reveal a significant role for the competitor's antibiotic as stimulator of own secondary metabolite production. Furthermore, we observed that V. coralliilyticus detoxifies holomycin by sulphur-methylation. Results presented here indicate that ecological competition in Vibrionaceae is mediated by, and a cue for, antibiotic secondary metabolite production.

Holomycin, an Antibiotic Secondary Metabolite, Is Required for Biofilm Formation by the Native Producer Photobacterium galatheae S2753.

While the effects of antibiotics on microorganisms are widely studied, it remains less well understood how antibiotics affect the physiology of the native producing organisms. Here, using a marine bacterium, Photobacterium galatheae S2753, that produces the antibiotic holomycin, we generated a holomycin-deficient strain by in-frame deletion of hlmE, the core gene responsible for holomycin production. Mass spectrometry analysis of cell extracts confirmed that the ΔhlmE strain did not produce holomycin and that the mutant was devoid of antibacterial activity. Biofilm formation of the ΔhlmE strain was significantly reduced compared to that of wild-type S2753 and was restored in an hlmE complementary mutant. Consistent with this, exogenous holomycin, but not its dimethylated and less antibacterial derivative, S,S'-dimethyl holomycin, restored the biofilm formation of the ΔhlmE strain. Furthermore, zinc starvation was found to be essential for both holomycin production and biofilm formation of S2753, although the molecular mechanism remains elusive. Collectively, these data suggest that holomycin promotes biofilm formation of S2753 via its ene-disulfide group. Lastly, the addition of holomycin at subinhibitory concentrations also enhanced the biofilms of four other Vibrionaceae strains. P. galatheae likely gains an ecological advantage from producing holomycin as both an antibiotic and a biofilm stimulator, which facilitates nutrition acquisition and protects P. galatheae from environmental stresses. Studying the function of antibiotic compounds in the native producer will shed light on their roles in nature and could point to novel bioprospecting strategies.IMPORTANCE Despite the societal impact of antibiotics, their ecological functions remain elusive and have mostly been studied by exposing nonproducing bacteria to subinhibitory concentrations. Here, we studied the effects of the antibiotic holomycin on its native producer, Photobacterium galatheae S2753, a Vibrionaceae bacterium. Holomycin provides a distinct advantage to S2753 both as an antibiotic and by enhancing biofilm formation in the producer. Vibrionaceae species successfully thrive in global marine ecosystems, where they play critical ecological roles as free-living, symbiotic, or pathogenic bacteria. Genome mining has demonstrated that many have the potential to produce several bioactive compounds, including P. galatheae To unravel the contribution of the microbial metabolites to the development of marine microbial ecosystems, better insight into the function of these compounds in the producing organisms is needed. Our finding provides a model to pursue this and highlights the ecological importance of antibiotics to the fitness of the producing organisms.

Production of the antimicrobial compound tetrabromopyrrole and the Pseudomonas quinolone system precursor, 2-heptyl-4-quinolone, by a novel marine species Pseudoalteromonas galatheae sp. nov.

Novel antimicrobials are urgently needed due to the rapid spread of antibiotic resistant bacteria. In a genome-wide analysis of Pseudoalteromonas strains, one strain (S4498) was noticed due to its potent antibiotic activity. It did not produce the yellow antimicrobial pigment bromoalterochromide, which was produced by several related type strains with which it shared less than 95% average nucleotide identity. Also, it produced a sweet-smelling volatile not observed from other strains. Mining the genome of strain S4498 using the secondary metabolite prediction tool antiSMASH led to eight biosynthetic gene clusters with no homology to known compounds, and synteny analyses revealed that the yellow pigment bromoalterochromide was likely lost during evolution. Metabolome profiling of strain S4498 using HPLC-HRMS analyses revealed marked differences to the type strains. In particular, a series of quinolones known as pseudanes were identified and verified by NMR. The characteristic odor of the strain was linked to the pseudanes. The highly halogenated compound tetrabromopyrrole was detected as the major antibacterial component by bioassay-guided fractionation. Taken together, the polyphasic analysis demonstrates that strain S4498 belongs to a novel species within the genus Pseudoalteromonas, and we propose the name Pseudoalteromonas galatheae sp. nov. (type strain S4498T = NCIMB 15250T = LMG 31599T).

New azaphilones from Aspergillus neoglaber.

Three new azaphilones, sassafrin E (1), sassafrin F (2), and sassafrinamine A (3), were isolated from the filamentous fungus Aspergillus neoglaber. The structures of the compounds were determined by nuclear magnetic resonance spectroscopy, and were found to be novel analogues of two already known compound classes; sassafrins and berkchaetoazaphilones. Sassafrin E and F were both oxygen containing, while sassafrinamine A additionally contained a nitrogen atom, originating from an aminoethanol moiety, as well as extensive conjugation resulting in an intense purple colour of the pure compound. The structure of sassafrin E was further confirmed using deuterium exchange experiments coupled with high-resolution tandem mass spectrometry.

Acurin A, a novel hybrid compound, biosynthesized by individually translated PKS- and NRPS-encoding genes in Aspergillus aculeatus.

This work presents the identification and proposed biosynthetic pathway for a compound of mixed polyketide-nonribosomal peptide origin that we named acurin A. The compound was isolated from an extract of the filamentous fungus Aspergillus aculeatus, and its core structure resemble that of the mycotoxin fusarin C produced by several Fusarium species. Based on bioinformatics in combination with RT-qPCR experiments and gene-deletion analysis, we identified a biosynthetic gene cluster (BGC) in A. aculeatus responsible for the biosynthesis of acurin A. Moreover, we were able to show that a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) enzyme separately encoded by this BGC are responsible for the synthesis of the PK-NRP compound, acurin A, core structure. In comparison, the production of fusarin C is reported to be facilitated by a linked PKS-NRPS hybrid enzyme. Phylogenetic analyses suggest the PKS and NRPS in A. aculeatus resulted from a recent fission of an ancestral hybrid enzyme followed by gene duplication. In addition to the PKS- and NRPS-encoding genes of acurin A, we show that six other genes are influencing the biosynthesis including a regulatory transcription factor. Altogether, we have demonstrated the involvement of eight genes in the biosynthesis of acurin A, including an in-cluster transcription factor. This study highlights the biosynthetic capacity of A. aculeatus and serves as an example of how the CRISPR/Cas9 system can be exploited for the construction of fungal strains that can be readily engineered.

Mass Spectrometry Guided Discovery and Design of Novel Asperphenamate Analogs From Penicillium astrolabium Reveals an Extraordinary NRPS Flexibility.

Asperphenamate is a small peptide natural product that has gained much interest due to its antitumor activity. In the recent years numerous bioactive synthetic asperphenamate analogs have been reported, whereas only a handful of natural analogs either of microbial or plant origin has been discovered. Herein we describe a UHPLC-HRMS/MS and amino acid supplement approach for discovery and design of novel asperphenamate analogs. Chemical analysis of Penicillium astrolabium, a prolific producer of asperphenamate, revealed three previously described and two novel asperphenamate analogs produced in significant amounts, suggesting a potential for biosynthesis of further asperphenamate analogs by varying the amino acid availability. Subsequent growth on proteogenic and non-proteogenic amino acid enriched media, revealed a series of novel asperphenamate analogs, including single or double amino acid exchange, as well as benzoic acid exchange for nicotinic acid, with the latter observed from a natural source for the first time. In total, 22 new asperphenamate analogs were characterized by HRMS/MS, with one additionally confirmed by isolation and NMR structure elucidation. This study indicates an extraordinary nonribosomal peptide synthetase (NRPS) flexibility based on substrate availability, and therefore the potential for manipulating and designing novel peptide natural products in filamentous fungi.

The Antibiotic Andrimid Produced by Vibrio coralliilyticus Increases Expression of Biosynthetic Gene Clusters and Antibiotic Production in Photobacterium galatheae.

The development and spread of multidrug resistant pathogens have reinforced the urgency to find novel natural products with antibiotic activity. In bacteria, orphan biosynthetic gene clusters (BGCs) far outnumber the BGCs for which chemistry is known, possibly because they are transcriptionally silent under laboratory conditions. A strategy to trigger the production of this biosynthetic potential is to challenge the microorganism with low concentrations of antibiotics, and by using a Burkholderia genetic reporter strain (Seyedsayamdost, Proc Natl Acad Sci 111:7266-7271), we found BGC unsilencing activity for the antimicrobial andrimid, produced by the marine bacterium Vibrio coralliilyticus. Next, we challenged another marine Vibrionaceae, Photobacterium galatheae, carrier of seven orphan BGCs with sub-inhibitory concentrations of andrimid. A combined approach of transcriptional and chemical measurements of andrimid-treated P. galatheae cultures revealed a 10-fold upregulation of an orphan BGC and, amongst others, a 1.6-2.2-fold upregulation of the gene encoding the core enzyme for biosynthesis of holomycin. Also, addition of andrimid caused an increase, based on UV-Vis peak area, of 4-fold in production of the antibiotic holomycin. Transcriptional measurements of stress response related genes in P. galatheae showed a co-occurrence of increased transcript levels of rpoS (general stress response) and andrimid induced holomycin overproduction, while in trimethoprim treated cultures attenuation of holomycin production coincided with a transcriptional increase of recA (SOS stress response). This study shows that using antimicrobial compounds as activators of secondary metabolism can be a useful strategy in eliciting biosynthetic gene clusters and facilitate natural product discovery. Potentially, such interactions could also have ecological relevant implications.

Atrorosins: a new subgroup of Monascus pigments from Talaromyces atroroseus.

A new series of azaphilone pigments named atrorosins have been isolated from the filamentous fungus Talaromyces atroroseus. Atrorosins have a similar azaphilone scaffold as the orange Monascus pigment PP-O, with a carboxylic acid group at C-1, but are unique by their incorporation of amino acids into the isochromene system. Despite that the atrorosin precursor PP-O, during fermentation, was initially produced as two isomers (3:2, cis:trans ratio), the atrorosins were surprisingly almost exclusively (99.5%) produced as the cis-form, possibly due to steric interactions with the incorporated amino acid. When grown on complex media, a whole range of atrorosins is produced, whereas individual atrorosins can be produced selectively during fermentation by supplementing with the desired primary amine-containing compound.

Unique processes yielding pure azaphilones in Talaromyces atroroseus.

Azaphilones are a class of fungal pigments, reported mostly in association with Monascus species. In Asian countries, they are used as food colourants under the name of "red yeast rice" and their production process is well described. One major limitation of current production techniques of azaphilones is that they always occur in a mixture of yellow, orange and red pigments. These mixtures are difficult to control and to quantify. This study has established a controlled and reproducible cultivation protocol to selectively tailor production of individual pigments during a submerged fermentation using another fungal species capable of producing azaphilone pigments, Talaromyces atroroseus, using single amino acids as the sole nitrogen source. The produced azaphilone pigments are called atrorosins and are amino acid derivatives of the known azaphilone pigment Penicillium purpurogenum-orange (PP-O), with the amino acid used as nitrogen source incorporated into the core skeleton of the azaphilone. This strategy was successfully demonstrated using 18 proteinogenic amino acids and the non-proteinogenic amino acid ornithine. Two cultivation methods for production of the pure serine derivative (atrorosin S) have been further developed, with yields of 0.9 g/L being obtained. Yielding pure atrorosins through switching from KNO3 to single amino acids as nitrogen source allows for considerably easier downstream processing and thus further enhances the commercial relevance of azaphilone producing fungal cell factories.

Genes Linked to Production of Secondary Metabolites in Talaromyces atroroseus Revealed Using CRISPR-Cas9.

The full potential of fungal secondary metabolism has until recently been impeded by the lack of universal genetic tools for most species. However, the emergence of several CRISPR-Cas9-based genome editing systems adapted for several genera of filamentous fungi have now opened the doors for future efforts in discovery of novel natural products and elucidation and engineering of their biosynthetic pathways in fungi where no genetic tools are in place. So far, most studies have focused on demonstrating the performance of CRISPR-Cas9 in various fungal model species, and recently we presented a versatile CRISPR-Cas9 system that can be successfully applied in several diverse Aspergillus species. Here we take it one step further and show that our system can be used also in a phylogenetically distinct and largely unexplored species from the genus of Talaromyces. Specifically, we exploit CRISPR-Cas9-based genome editing to identify a new gene in T. atroroseus responsible for production of polyketide-nonribosomal peptide hybrid products, hence, linking fungal secondary metabolites to their genetic origin in a species where no genetic engineering has previously been performed.

Linker Flexibility Facilitates Module Exchange in Fungal Hybrid PKS-NRPS Engineering.

Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) each give rise to a vast array of complex bioactive molecules with further complexity added by the existence of natural PKS-NRPS fusions. Rational genetic engineering for the production of natural product derivatives is desirable for the purpose of incorporating new functionalities into pre-existing molecules, or for optimization of known bioactivities. We sought to expand the range of natural product diversity by combining modules of PKS-NRPS hybrids from different hosts, hereby producing novel synthetic natural products. We succeeded in the construction of a functional cross-species chimeric PKS-NRPS expressed in Aspergillus nidulans. Module swapping of the two PKS-NRPS natural hybrids CcsA from Aspergillus clavatus involved in the biosynthesis of cytochalasin E and related Syn2 from rice plant pathogen Magnaporthe oryzae lead to production of novel hybrid products, demonstrating that the rational re-design of these fungal natural product enzymes is feasible. We also report the structure of four novel pseudo pre-cytochalasin intermediates, niduclavin and niduporthin along with the chimeric compounds niduchimaeralin A and B, all indicating that PKS-NRPS activity alone is insufficient for proper assembly of the cytochalasin core structure. Future success in the field of biocombinatorial synthesis of hybrid polyketide-nonribosomal peptides relies on the understanding of the fundamental mechanisms of inter-modular polyketide chain transfer. Therefore, we expressed several PKS-NRPS linker-modified variants. Intriguingly, the linker anatomy is less complex than expected, as these variants displayed great tolerance with regards to content and length, showing a hitherto unreported flexibility in PKS-NRPS hybrids, with great potential for synthetic biology-driven biocombinatorial chemistry.