The isoprenyl chain length of coenzyme Q mediates the nutritional resistance of fungi to amoeba predation.

Amoebae are environmental predators feeding on bacteria, fungi, and other eukaryotic microbes. Predatory interactions alter microbial communities and impose selective pressure toward phagocytic resistance or escape which may, in turn, foster virulence attributes. The ubiquitous fungivorous amoeba Protostelium aurantium has a wide prey spectrum in the fungal kingdom but discriminates against members of the Saccharomyces clade, such as Saccharomyces cerevisiae and Candida glabrata. Here, we show that this prey discrimination among fungi is solely based on the presence of ubiquinone as an essential cofactor for the predator. While the amoeba readily fed on fungi with CoQ presenting longer isoprenyl side chain variants CoQ8-10, such as those from the Candida clade, it failed to proliferate on those with shorter CoQ variants, specifically from the Saccharomyces clade (CoQ6). Supplementing non-edible yeast with CoQ9 or CoQ10 rescued the growth of P. aurantium, highlighting the importance of a long isoprenyl side chain. Heterologous biosynthesis of CoQ9 in S. cerevisiae by introducing genes responsible for CoQ9 production from the evolutionary more basic Yarrowia lipolytica complemented the function of the native CoQ6. The results suggest that the use of CoQ6 among members of the Saccharomyces clade might have originated as a predatory escape strategy in fungal lineages and could be retained in organisms that were able to thrive by fermentation. IMPORTANCE: Ubiquinones (CoQ) are universal electron carriers in the respiratory chain of all aerobic bacteria and eukaryotes. Usually 8-10 isoprenyl units ensure their localization within the lipid bilayer. Members of the Saccharomyces clade among fungi are unique in using only 6. The reason for this is unclear. Here we provide evidence that the use of CoQ6 efficiently protects these fungi from predation by the ubiquitous fungivorous amoeba Protostelium aurantium which lacks its own biosynthetic pathway for this vitamin. The amoebae were starving on a diet of CoQ6 yeasts which could be complemented by either the addition of longer CoQs or the genetic engineering of a CoQ9 biosynthetic pathway.

The spatial organization of sphingofungin biosynthesis in Aspergillus fumigatus and its cross-interaction with sphingolipid metabolism.

Sphingofungins are sphinganine analog mycotoxins acting as inhibitors of serine palmitoyl transferases, enzymes responsible for the first step in the sphingolipid biosynthesis. Eukaryotic cells are highly organized with various structures and organelles to facilitate cellular processes and chemical reactions, including the ones occurring as part of the secondary metabolism. We studied how sphingofungin biosynthesis is compartmentalized in the human-pathogenic fungus Aspergillus fumigatus, and we observed that it takes place in the endoplasmic reticulum (ER), ER-derived vesicles, and the cytosol. This implies that sphingofungin and sphingolipid biosynthesis colocalize to some extent. Automated analysis of confocal microscopy images confirmed the colocalization of the fluorescent proteins. Moreover, we demonstrated that the cluster-associated aminotransferase (SphA) and 3-ketoreductase (SphF) play a bifunctional role, supporting sphingolipid biosynthesis, and thereby antagonizing the toxic effects caused by sphingofungin production.IMPORTANCEA balanced sphingolipid homeostasis is critical for the proper functioning of eukaryotic cells. To this end, sphingolipid inhibitors have therapeutic potential against diseases related to the deregulation of sphingolipid balance. In addition, some of them have significant antifungal activity, suggesting that sphingolipid inhibitors-producing fungi have evolved mechanisms to escape self-poisoning. Here, we propose a novel self-defense mechanism, with cluster-associated genes coding for enzymes that play a dual role, being involved in both sphingofungin and sphingolipid production.

Engineered living materials for the conversion of a low-cost food-grade precursor to a high-value flavonoid.

Microbial biofactories allow the upscaled production of high-value compounds in biotechnological processes. This is particularly advantageous for compounds like flavonoids that promote better health through their antioxidant, anti-bacterial, anti-cancer and other beneficial effects but are produced in small quantities in their natural plant-based hosts. Bacteria like E. coli have been genetically modified with enzyme cascades to produce flavonoids like naringenin and pinocembrin from coumaric or cinnamic acid. Despite advancements in yield optimization, the production of these compounds still involves high costs associated with their biosynthesis, purification, storage and transport. An alternative production strategy could involve the direct delivery of the microbial biofactories to the body. In such a strategy, ensuring biocontainment of the engineered microbes in the body and controlling production rates are major challenges. In this study, these two aspects are addressed by developing engineered living materials (ELMs) consisting of probiotic microbial biofactories encapsulated in biocompatible hydrogels. Engineered probiotic E. coli Nissle 1917 able to efficiently convert cinnamic acid into pinocembrin were encapsulated in poly(vinyl alcohol)-based hydrogels. The biofactories are contained in the hydrogels for a month and remain metabolically active during this time. Control over production levels is achieved by the containment inside the material, which regulates bacteria growth, and by the amount of cinnamic acid in the medium.

Advances in Synthetic Biology of Fungi and Contributions to the Discovery of New Molecules.

The sequencing of fungal genomes is becoming increasingly accessible, with a wealth of data already available. In parallel, the prediction of the putative biosynthetic pathways responsible for the synthesis of potential new natural products is also increasing. The difficulty of translating computational analyses into available compounds is becoming evident, slowing down a process that was thought to be faster with the advent of the genomic era. Advances in gene techniques made it possible to genetically modify a wider range of organisms, including fungi typically considered recalcitrant to DNA manipulation. However, the possibility of screening many gene cluster products for new activities in a high-throughput manner remains unfeasible. Nonetheless, some updates on the synthetic biology of fungi could provide interesting insights that could help to achieve this goal in the future.

Scale-up of an amoeba-based process for the production of the cannabinoid precursor olivetolic acid.

BACKGROUND: The availability of new biological platform organisms to get access to innovative products and processes is fundamental for the progress in biotechnology and bioeconomy. The amoeba Dictyostelium discoideum represents a novel host system that has recently been employed for both the discovery of new natural products and as a cell factory for the production of bioactive compounds such as phytochemicals. However, an essential parameter to evaluate the potential of a new host system is the demonstration of its scalability to allow industrial applicability. Here, we aimed to develop a bioprocess for the production of olivetolic acid, the main precursor of cannabinoids synthesized by a recently engineered D. discoideum strain. RESULTS: In this study, a sophisticated approach is described to scale-up an amoeba-based polyketide production process in stirred tank bioreactors. Due to the shear sensitivity of the cell wall lacking amoebae, the maximum local energy dissipation rate (εmax) was selected as a measure for the hydromechanical stress level among different scales. By performing 1.6-L scale batch fermentations with different stress conditions, we determined a maximum tolerable εmax of 3.9 W/kg for D. discoideum. Further, we used this parameter as scale-up criterion to develop a bioprocess for olivetolic acid production starting from a 7-L stirred tank reactor to the industrially relevant 300-L scale with a product concentration of 4.8 µg/L, a productivity of 0.04 µg/L/h and a yield of 0.56 µg/g glucose. CONCLUSION: We developed a robust and reliable scale-up strategy for amoeba-based bioprocesses and evaluated its applicability for the production of the cannabinoid precursor olivetolic acid. By determining the maximum tolerable hydromechanical stress level for D. discoideum, we were able to scale-up the process from shake flasks to the 300-L stirred tank reactor without any yield reduction from cell shearing. Hence, we showed the scalability and biotechnological exploitation of amoeba-based processes that can provide a reasonable alternative to chemical syntheses or extractions of phytochemicals from plant biomass.

Yellow polyketide pigment suppresses premature hatching in social amoeba.

Low-molecular-weight natural products from microbes are indispensable in the development of potent drugs. However, their biological roles within an ecological context often remain elusive. Here, we shed light on natural products from eukaryotic microorganisms that have the ability to transition from single cells to multicellular organisms: the social amoebae. These eukaryotes harbor a large number of polyketide biosynthetic genes in their genomes, yet virtually none of the corresponding products can be isolated or characterized. Using complementary molecular biology approaches, including CRISPR-Cas9, we generated polyketide synthase (pks5) inactivation and overproduction strains of the social amoeba Dictyostelium discoideum. Differential, untargeted metabolomics of wild-type versus mutant fruiting bodies allowed us to pinpoint candidate metabolites derived from the amoebal PKS5. Extrachromosomal expression of the respective gene led to the identification of a yellow polyunsaturated fatty acid. Analysis of the temporospatial production pattern of this compound in conjunction with detailed bioactivity studies revealed the polyketide to be a spore germination suppressor.

Desaturation of the Sphingofungin Polyketide Tail Results in Increased Serine Palmitoyltransferase Inhibition.

Serine palmitoyltransferase catalyzes the first step of the sphingolipid biosynthesis. Recently, sphingolipid homeostasis has been connected to several human diseases, making serine palmitoyltransferases an interesting therapeutic target. Known and efficient serine palmitoyltransferase-inhibitors are sphingofungins, a group of natural products isolated from fungi. To further characterize newly isolated sphingofungins, we designed an easy to use colorimetric serine palmitoyltransferase activity assay using FadD, which can be performed in 96-well plates. Because sphingofungins exert antifungal activitiy as well, we compared the in vitro assay results with an in vivo growth assay using Saccharomyces cerevisiae. The reported experiments showed differences among the assayed sphingofungins, highlighting an increase of activity based on the saturation levels of the polyketide tail. IMPORTANCE Targeting the cellular sphingolipid metabolism is often discussed as a potential approach to treat associated human diseases such as cancer and Alzheimer's disease. Alternatively, it is also a possible target for the development of antifungal compounds, which are direly needed. A central role is played by the serine palmitoyltransferase, which catalyzes the initial and rate limiting step of sphingolipid de novo synthesis and, as such, the development of inhibitory compounds for this enzyme is of interest. Our work here established an alternative approach for determining the activity of serine palmitoyltransferase adding another tool for the validation of its inhibition. We also determined the effect of different modifications to sphingofungins on their inhibitory activity against serine palmitoyltransferase, revealing important differences on said activity against enzymes of bacterial and fungal origin.

Intrinsic Ability of the ß-Oxidation Pathway To Produce Bioactive Styrylpyrones.

Naturally occurring α-pyrones with biological activities are mostly synthesised by polyketide synthases (PKSs) via iterative decarboxylative Claisen condensation steps. Remarkably, we found that some enzymes related to the fatty acid ß-oxidation pathway in Escherichia coli, namely the CoA ligase FadD and the thiolases FadA and FadI, can synthesise styrylpyrones with phenylpropionic acids in vivo. The two thiolases directly utilise acetyl-CoA as an extender unit for carbon-chain elongation through a non-decarboxylative Claisen condensation, thus making the overall reaction more efficient in terms of carbon and energy consumption. Moreover, using a cell-free approach, different styrylpyrones were synthesised in vitro. Finally, targeted feeding experiments led to the detection of styrylpyrones in other species, demonstrating that the intrinsic ability of the ß-oxidation pathway allows for the synthesis of such molecules in bacteria, revealing an important biological feature hitherto neglected.

Chemical Diversity and Biosynthesis of Drimane-Type Sesquiterpenes in the Fungal Kingdom.

Drimane-type sesquiterpenes are a class of compounds produced by a wide range of organisms, initially isolated and characterized in plants. Meanwhile, in the past 20-30 years, a large number of novel structures from many divergent fungi have been elucidated. Recently, the biosynthesis of drimane-type sesquiterpenes and their esters has been explained in two filamentous fungi, namely Aspergillus oryzae and Aspergillus calidoustus, disclosing the basic biosynthetic principles needed to identify similar pathways in the fungal kingdom.

Engineering the amoeba Dictyostelium discoideum for biosynthesis of a cannabinoid precursor and other polyketides.

Aromatic polyketides are natural polyphenolic compounds with a broad spectrum of pharmacological activities. Production of those metabolites in the model organisms Escherichia coli and Saccharomyces cerevisiae has been limited by the extensive cellular engineering needed for the coordinated biosynthesis of polyketides and their precursors. In contrast, the amoeba Dictyostelium discoideum is a native producer of secondary metabolites and harbors a wide, but largely unexplored, repertoire of genes for the biosynthesis of polyketides and terpenoids. Here we present D. discoideum as an advantageous chassis for the production of aromatic polyketides. By expressing its native and cognate plant polyketide synthase genes in D. discoideum, we demonstrate production of phlorocaprophenone, methyl-olivetol, resveratrol and olivetolic acid (OA), which is the central intermediate in the biosynthesis of cannabinoids. To facilitate OA synthesis, we further engineered an amoeba/plant inter-kingdom hybrid enzyme that produced OA from primary metabolites in two enzymatic steps, providing a shortcut in a synthetic cannabinoid pathway using the D. discoideum host system.

Biosynthesis of the Sphingolipid Inhibitors Sphingofungins in Filamentous Fungi Requires Aminomalonate as a Metabolic Precursor.

Sphingofungins belong to a group of structurally related sphingolipid inhibitors produced by fungi, which specifically inhibit serine palmitoyl transferases, enzymes catalyzing the initial step during sphingolipid biosynthesis. Sphingolipids are integral parts of the eukaryotic cell membrane, and disturbances in their homeostasis have been linked to various human diseases. It has been suggested that external interventions, via sphingolipid inhibitors, may represent a promising approach for alternative therapies. Here, we identified and elucidated the biosynthetic gene cluster responsible for the biosynthesis of sphingofungins B, C, and D in Aspergillus fumigatus. Moreover, in vitro analyses have shown that sphingofungin biosynthesis starts with the condensation of a C18 polyketide with the uncommon substrate aminomalonate. Furthermore, the investigations on sphingofungin E and F produced by Paecilomyces variotii pointed out that different aminomalonate derivatives are used as substrates for those chemical variants. This research boosts knowledge on the general biosynthesis of sphingolipid inhibitors in fungi.

Time-resolved multiparameter analytics on a cell-free production platform for acyl-CoA precursors.

Cell-free biosynthesis is emerging as a very attractive alternative for the production of market-relevant molecules. The free combination of enzymes, regardless of where they are isolated from, raises the possibility to build more efficient synthetic routes but at the same time leads to higher complexity regarding the analysis of the different enzymatic steps. Here we present an analytical method for the real-time analysis of acyl-CoA blocks forming and consuming during multi-step catalyses. We focused on malonyl-Coenzyme A and acetyl-CoA, which are the most used acyl-CoA units for carbon chain elongations. By employing capillary electrophoresis, we could detect the decrease of educts and the formation of products in a time-resolved fashion.

Biosynthesis of Fungal Drimane-Type Sesquiterpene Esters.

Drimane-type sesquiterpenes exhibit various biological activities and are widely present in eukaryotes. Here, we completely elucidated the biosynthetic pathway of the drimane-type sesquiterpene esters isolated from Aspergillus calidoustus and we discovered that it involves a drimenol cyclase having the same catalytic function previously only reported in plants. Moreover, since many fungal drimenol derivatives possess a γ-butyrolactone ring, we clarified the functions of the cluster-associated cytochrome P450 and FAD-binding oxidoreductase discovering that these two enzymes are solely responsible for the formation of those structures. Furthermore, swapping of the enoyl reductase domain in the identified polyketide synthase led to the production of metabolites containing various polyketide chains with different levels of saturation. These findings have deepened our understanding of how fungi synthesize drimane-type sesquiterpenes and the corresponding esters.

Transcriptional Control of the Production of Aspergillus fumigatus Conidia-Borne Secondary Metabolite Fumiquinazoline C Important for Phagocytosis Protection.

Aspergillus fumigatus produces diverse secondary metabolites whose biological functions and regulation remain to be understood. Despite the importance of the conidia for this fungus, the role of the conidia-born metabolite fumiquinazoline C (FqC) is unclear. Here, we describe a dual function of the cell-wall integrity pathway in regulating FqC biosynthesis dictated by the MAPK kinase MpkA, which phosphorylates one of the nonribosomal peptide synthetases enzymes of the cluster (FmqC), and the transcription factor RlmA, which directly regulates the expression of fmq genes. Another level of crosstalk between the FqC regulation and the cell physiology is described since the deletion of the stress-responsive transcription factor sebA provokes derepression of the fmq cluster and overproduction of FqC. Thus, we describe a mechanism by which A. fumigatus controls FqC biosynthesis orchestrated by MpkA-RlmA and SebA and hence enabling survival and adaptation to the environmental niche, given that FqC is a deterrent of ameba predation.

Self-Protection against the Sphingolipid Biosynthesis Inhibitor Fumonisin B1 Is Conferred by a FUM Cluster-Encoded Ceramide Synthase.

Fumonisin (FB) mycotoxins produced by species of the genus Fusarium detrimentally affect human and animal health upon consumption, due to the inhibition of ceramide synthase. In the present work, we set out to identify mechanisms of self-protection employed by the FB1 producer Fusarium verticillioides FB1 biosynthesis was shown to be compartmentalized, and two cluster-encoded self-protection mechanisms were identified. First, the ATP-binding cassette transporter Fum19 acts as a repressor of the FUM gene cluster. Appropriately, FUM19 deletion and overexpression increased and decreased, respectively, the levels of intracellular and secreted FB1 Second, the cluster genes FUM17 and FUM18 were shown to be two of five ceramide synthase homologs in Fusarium verticillioides, grouping into the two clades CS-I and CS-II in a phylogenetic analysis. The ability of FUM18 to fully complement the yeast ceramide synthase null mutant LAG1/LAC1 demonstrated its functionality, while coexpression of FUM17 and CER3 partially complemented, likely via heterodimer formation. Cell viability assays revealed that Fum18 contributes to the fungal self-protection against FB1 and increases resistance by providing FUM cluster-encoded ceramide synthase activity.IMPORTANCE The biosynthesis of fungal natural products is highly regulated not only in terms of transcription and translation but also regarding the cellular localization of the biosynthetic pathway. In all eukaryotes, the endoplasmic reticulum (ER) is involved in the production of organelles, which are subject to cellular traffic or secretion. Here, we show that in Fusarium verticillioides, early steps in fumonisin production take place in the ER, together with ceramide biosynthesis, which is targeted by the mycotoxin. A first level of self-protection is given by the presence of a FUM cluster-encoded ceramide synthase, Fum18, hitherto uncharacterized. In addition, the final fumonisin biosynthetic step occurs in the cytosol and is thereby spatially separate from the fungal ceramide synthases. We suggest that these strategies help the fungus to avoid self-poisoning during mycotoxin production.

Rational Design of Flavonoid Production Routes Using Combinatorial and Precursor-Directed Biosynthesis.

Combinatorial biosynthesis has great potential for designing synthetic circuits and amplifying the production of new active compounds. Studies on multienzyme cascades are extremely useful for improving our knowledge on enzymatic catalysis. In particular, the elucidation of enzyme substrate promiscuity can be potentially used for bioretrosynthetic approaches, leading to the design of alternative and more convenient routes to produce relevant molecules. In this perspective, plant-derived polyketides are extremely adaptable to those synthetic biological applications. Here, we present a combination of an in vitro CoA ligase activity assay coupled with a bacterial multigene expression system that leads to precursor-directed biosynthesis of 21 flavonoid derivatives. When the vast knowledge from protein databases is exploited, the herein presented procedure can be easily repeated with additional plant-derived polyketides. Lastly, we report an efficient in vivo expression system that can be further exploited to heterologously express pathways not necessarily related to plant polyketide synthases.

Thermomagneto-Responsive Smart Biocatalysts for Malonyl-Coenzyme A Synthesis.

Smart biocatalysts, in which enzymes are conjugated to stimuli-responsive polymers, have gained considerable attention because of their catalytic switchability and recyclability. Although many systems have been developed, they require separate laboratory techniques for their recovery, making them unsuitable for many practical applications. To address these issues, we designed a thermomagneto-responsive biocatalyst by immobilizing an enzyme on the terminal of thermo-responsive polymer brushes tethered on magnetic nanoparticle (NP) clusters. The concept is demonstrated by a system consisting of iron oxide NPs, poly(N-isopropyl-acrylamide), and a malonyl-Coenzyme A synthetase (MatB). By using free malonate and coenzyme A (CoA), the designed catalyst exhibits adequate activity for the production of malonyl-CoA. Thanks to the use of a magnetic NP cluster, whose magnetic moment is high, this system is fully recoverable under the magnetic field at above 32 °C because of the collapse of the thermo-responsive polymer shell in the clusters. In addition, the recycled catalyst maintains moderate activity even after three cycles, and it also shows excellent catalytic switchability, that is, negligible catalytic activity at 25 °C because of the blockage of the active sites of the enzyme by the extended hydrophilic polymer chains but great catalytic activity at a temperatures above the lower critical solution temperature at which the enzymes are exposed to the reaction medium because of the thermo-responsive contraction of polymer chains. Because the azide functionality in our system can be easily functionalized depending upon our need, such catalytically switchable, fully recoverable, and recyclable multiresponsive catalytic systems can be of high relevance for other cell-free biosynthetic approaches.

The fungal CCAAT-binding complex and HapX display highly variable but evolutionary conserved synergetic promoter-specific DNA recognition.

To sustain iron homeostasis, microorganisms have evolved fine-tuned mechanisms for uptake, storage and detoxification of the essential metal iron. In the human pathogen Aspergillus fumigatus, the fungal-specific bZIP-type transcription factor HapX coordinates adaption to both iron starvation and iron excess and is thereby crucial for virulence. Previous studies indicated that a HapX homodimer interacts with the CCAAT-binding complex (CBC) to cooperatively bind bipartite DNA motifs; however, the mode of HapX-DNA recognition had not been resolved. Here, combination of in vivo (genetics and ChIP-seq), in vitro (surface plasmon resonance) and phylogenetic analyses identified an astonishing plasticity of CBC:HapX:DNA interaction. DNA motifs recognized by the CBC:HapX protein complex comprise a bipartite DNA binding site 5'-CSAATN12RWT-3' and an additional 5'-TKAN-3' motif positioned 11-23 bp downstream of the CCAAT motif, i.e. occasionally overlapping the 3'-end of the bipartite binding site. Phylogenetic comparison taking advantage of 20 resolved Aspergillus species genomes revealed that DNA recognition by the CBC:HapX complex shows promoter-specific cross-species conservation rather than regulon-specific conservation. Moreover, we show that CBC:HapX interaction is absolutely required for all known functions of HapX. The plasticity of the CBC:HapX:DNA interaction permits fine tuning of CBC:HapX binding specificities that could support adaptation of pathogens to their host niches.

Targeted induction of a silent fungal gene cluster encoding the bacteria-specific germination inhibitor fumigermin.

Microorganisms produce numerous secondary metabolites (SMs) with various biological activities. Many of their encoding gene clusters are silent under standard laboratory conditions because for their activation they need the ecological context, such as the presence of other microorganisms. The true ecological function of most SMs remains obscure, but understanding of both the activation of silent gene clusters and the ecological function of the produced compounds is of importance to reveal functional interactions in microbiomes. Here, we report the identification of an as-yet uncharacterized silent gene cluster of the fungus Aspergillus fumigatus, which is activated by the bacterium Streptomyces rapamycinicus during the bacterial-fungal interaction. The resulting natural product is the novel fungal metabolite fumigermin, the biosynthesis of which requires the polyketide synthase FgnA. Fumigermin inhibits germination of spores of the inducing S. rapamycinicus, and thus helps the fungus to defend resources in the shared habitat against a bacterial competitor.