Non-canonical two-step biosynthesis of anti-oomycete indole alkaloids in Kickxellales

Background Fungi are prolific producers of bioactive small molecules of pharmaceutical or agricultural interest. The secondary metabolism of higher fungi (Dikarya) has been well-investigated which led to > 39,000 described compounds. However, natural product researchers scarcely drew attention to early-diverging fungi (Mucoro- and Zoopagomycota) as they are considered to rarely produce secondary metabolites. Indeed, only 15 compounds have as yet been isolated from the entire phylum of the Zoopagomycota. Results Here, we showcase eight species of the order Kickxellales (phylum Zoopagomycota) as potent producers of the indole-3-acetic acid (IAA)-derived compounds lindolins A and B. The compounds are produced both under laboratory conditions and in the natural soil habitat suggesting a specialized ecological function. Indeed, lindolin A is a selective agent against plant-pathogenic oomycetes such as Phytophthora sp. Lindolin biosynthesis was reconstituted in vitro and relies on the activity of two enzymes of dissimilar evolutionary origin: Whilst the IAA–CoA ligase LinA has evolved from fungal 4-coumaryl-CoA synthetases, the subsequently acting IAA-CoA:anthranilate N-indole-3-acetyltransferase LinB is a unique enzyme across all kingdoms of life. Conclusions This is the first report on bioactive secondary metabolites in the subphylum Kickxellomycotina and the first evidence for a non-clustered, two-step biosynthetic route of secondary metabolites in early-diverging fungi. Thus, the generally accepted “gene cluster hypothesis” for natural products needs to be reconsidered for early diverging fungi. Supplementary Information The online version contains supplementary material available at 10.1186/s40694-023-00166-x.

Saprotrophic kickxellomycetes are rarely isolated from nature, but preferably from rhizosphere, soil, humus, dung, and other organic material from dead plants and animals [2,15].Colonies usually grow slowly which is why they are quickly covered by other saprobe ascomycetes prior to isolation [15].With at least 167 isolated and ITS sequenced species, Coemansia is the major genus among the subphylum Kickxellomycotina [2].In contrast, the rarely isolated kickxellomycetes include Linderina species (with L. pennispora and L. macrosporum as the sole identified species) [16], Martensiomyces pterosporus [17] and Kickxella alabastrina [18,19].However, although they are easily cultivable representatives of the Zoopagomycota, neither their metabolic potential nor their ecological impact have been investigated yet.
We addressed this profound knowledge gap.Here, we report that L. pennispora, M. pterosporus, and six Coemansia species are producers of bioactive indole alkaloids.The indole-3-acetic acid (IAA)-derived anthranilic amides, lindolin A and B, are secreted to the culture supernatant and during growth in soil organic matter suggesting an ecological relevance.Indeed, lindolins possess moderate, but selective activity against the plantpathogenic oomycete Phytophthora megasperma whilst other antimicrobial, phyto-or cytotoxic side activities were not detected.The combined activities of the IAA-CoA ligase LinA and the unique IAA-CoA:anthranilate N-indole-3-acetyltransferase LinB catalyze in the NRP synthetase (NRPS)-independent amide formation of lindolins.The transferase LinB is a unique enzyme and highly conserved among all Kickxellales indicating lindolin biosynthesis as a chemotaxonomic marker of this prolific fungal order.

The secondary metabolism of the fungal division Zoopagomycota is underexplored
Initially, we screened the LOTUS natural product data bank for secondary metabolite (SM) producers among the EDF [20].As expected, higher fungi (Dikarya) have been intensively studied and dominated the database with approx.40,000 fungal compounds by far (Fig. 1A).However, EDF are hardly known to produce SMs as merely 1% of the fungal metabolites (459 compounds) are EDF-derived.The most prominent producers are species from the Mucoromycota (442 compounds), whilst in sum solely 17 metabolites have been isolated from the three major phyla Zoopagomycota, Blastocladiomycota and Chytridiomycota altogether (Fig. 1B).

Lindolins are produced in soil
Lindolins are preferably isolated from the supernatant of submerse cultures in L. pennispora, M. pterosporus and C. furcata, but are not detectable in mycelia, suggesting Fig. 1 Overview of known metabolites of early diverging fungi (EDF) and Zoopagomycota.A The metabolite count was extracted from all microbial kingdoms and fungal orders from the LOTUS database and was additionally manually inspected for EDF [20].B Metabolites from the fungal phylum Zoopagomycota are rare and include peptides [21][22][23], polyenes [24] and azoxyaromates [25].Note, that this survey is non-exhaustive as kingdom-wide common primary metabolites such as unmodified sesquiterpenes, fatty acids and their degradation products are not included that the compounds are actively secreted (Fig. 3A, B) Similarly, emerse cultures (agar plates) led to the production of lindolins (Fig. 3C).However, cultivation in flasks and on plates are highly artificial growth conditions and do not resemble the natural habitat.Though, L. pennispora, M. pterosporus and C. furcata were additionally cultivated in potting soil in presence of d-glucose to boost the fungal growth (Fig. 3D).Again, 4 and 5 were detectable in at least two of the three species indicating that lindolins are produced in nature by Kickxellales and may have an impact in modulating their ecological niche.

Lindolins are anti-oomycete compounds
We screened for a potential ecological function of lindolins with various biological assays.Lindolins are neither antibacterial compounds nor cytotoxic against mammalian cells (Additional file 26: Figure S23 and Additional file 27: Figure S24).As kickxellomycetes were isolated from grasslands [34] and fungal growth is positively correlated with a high carbon-to-nitrogen ratio [35], we studied the impact of lindolins on plant growth: Because lindolins share structural similarities to the major plant hormone auxin (indole-3-acetic acid, IAA, 3) [36], we specifically determined root growth inhibitory activity of the compounds using radish seedlings (Additional file 28: Figure S25).However, lindolins (4-5) neither induce root shortening as 3 nor impairs 3-mediated plant growth in general.Hence, at least on the tested seedlings, lindolins do not show phytotoxic activities.However, during our initial screening we recognized an activity of lindolins against the plant pathogenic oomycete Phytophthora megasperma, while plant pathogenic fungi (e.g.Fusarium graminearum) were unaffected (Additional file 29: Figure S26).4 showed moderate activity against oomycetes P. megasperma, (MIC 50 of 100 ± 11 µM) and-to a lower extend-Pythium macrosporum (MIC 50 215 ± 61 µM), whilst 5 did not inhibit oomycete growth (MIC 50 > 500 µM), suggesting that the 5ʹ-hydroxylation dramatically impairs bioactivity (Additional file 30: Figure S27).

In silico reconstruction of the lindolin biosynthetic pathway
In EDF, peptide compounds are usually formed by NRPSs as demonstrated for hexapeptides [9,37] and cyclopentapeptides [38] from M. alpina and postulated for the cyclopentapeptides from B. meristosporus [21].Commonly, small fungal amides such as fumarylalanine from the pathogenic ascomycete Aspergillus fumigatus [39] or benzodiazephinedione from Neosartorya fischeri [40] are synthesized by condensation of two (amino) acids using a bimodular NRPS.However, the genomes of L. pennispora and related Kickxellales species lack genes for bimodular NRPSs [2].Therefore, we postulated a coenzyme A (CoA)-mediated route for the production of lindolins (Fig. 4A).
The subsequent amidation of 3 with anthranilic acid (AA) requires two separate steps: (i) an activation of the chemically unreactive carboxylic acid of 3 by adenylation and subsequent thioesterification by a ligase and (ii) the final amidation of IAA with AA by a transferase (Fig. 4A).A similar mechanism has been shown for the avenanthramide biosynthesis in oat (Avena sativa) [48].Hereby, the 4-coumarate-CoA ligase 4CL activates 4-coumaric acid to 4-coumaryl-CoA, which in turn is amidated with 5-hydroxyanthranilic acid by the hydroxycinnamoyl-CoA:5-hydroxy anthranilate N-hydroxycinnamoyl transferase HHT1 to yield the final product avenanthramide A (Fig. 4B).We searched the genome for probable candidates using the 4CL genes from A. sativa and Arabidopsis thaliana as queries and identified 31 4CL-homologous genes, among them linA (DL89DRAFT_225601; e value 1 × 10 -101 ) (Table 1).In contrast, when we used the A. sativa HHT1 as query, 21 hits with e values of only > 1 × 10 -15 were obtained.However, when we compared the sequences pterosporus] showed the highest similarity (76% pairwise identity) and lowest bit-score (e value 0) (Table 1).
The most likely ligase gene linA and the most probable hht-like candidate gene linB from L. pennispora were  chosen for expressional profiling.Interestingly, both genes are co-expressed under lindolin producing conditions (Fig. 5A).

LinA is an indole-3-acetate-CoA ligase
The ligase gene linA was heterologously expressed in Escherichia coli, and the enzyme was purified as a C-terminal His 6 -tagged soluble fusion protein.We investigated the substrate specificity of LinA by measuring the released AMP by monitoring the depletion of NADH  Fig. 5 Expressional profiling of lin genes and enzyme activities of the ligase LinA and the transferase LinB.A Expressional profiling of linA and linB in L. pennispora grown in meat and PDB medium.The expression was referenced to growth in Aspergillus minimal medium and normalized against the housekeeping gene encoding the glyceraldehyde-3-phosphate dehydrogenase (gpdA).Both genes were co-expressed.B Coupled multi-enzyme assay to determine the substrate specificity of LinA.C Substrate profiling of LinA.All substrates were used at a final concentration of 1 mM.Water served as negative control.The assay was performed as described [49].D LinB activity assay.LinB was incubated with IAA-CoA and either AA to produce 4 or HAA to produce 5. Overlaid extracted ion chromatograms (EIC) were shown for 4 (m/z 293 [M−H] − , in red) and 5 (m/z 309 [M−H] − ), in blue), respectively.Assays without enzyme or without substrates were included as negative controls.Authentic standards of 4 and 5 served as positive controls with a well-established enzyme-coupled assay [49] (Fig. 5B, Additional file 33: Figure S29).LinA preferably uses IAA (3) as substrate followed by 2-ketoisovaleric acid, acetate and 2-keto-3-methylvaleric acid (Fig. 5C).The enzyme is highly specific for 3 as it does not convert chemically similar aryl acids such as 4-coumaric acid, caffeic acid or the IAA-analog indole-3-butyric acid (IBA).We determined optimal conditions at pH = 7.5 and ϑ = 35 °C (Additional file 34: Figure S30).Hence, we established LinA as an IAA-CoA ligase.

LinB is an indole-3-acetyl-CoA:anthranilate N-indole-3-acetyltransferase
The transferase LinB was similarly purified from recombinant E. coli as an N-terminally His 6 -tagged protein (Additional file 33: Figure S29).To characterize LinB, its potential substrate IAA-CoA was synthesized by a reported protocol [50].When LinB and IAA-CoA were incubated with the acceptor substrates anthranilic acid (AA) or 5-hydroxyanthranilic acid (HAA), formation of 4 or 5 was observed (Fig. 5D).Hence, LinB is sufficient for the production of either lindolin.Moreover, 5 is synthesized from HAA rather than by oxidation of 4.However, when both anthranilic substrates are equimolarly provided, AA is clearly preferred over HAA (Additional file 35: Figure S31).We determined a pH optimum (pH = 7.8) and a temperature optimum (ϑ = 30 °C) similar to that of LinA (Additional file 36: Figure S32) and assigned LinB as an IAA-CoA:anthranilate N-indole-3-acetyltransferase.− .Note, that the CoA ester leads to slightly broadened peaks as described elsewhere [51].E Enzyme-coupled determination of LinA activity in presence and absence of LinB

LinA and LinB are sufficient to produce lindolins in vitro
We tested the production of lindolins in a one-potreaction (Fig. 6A).Indeed, using both enzymes (LinA and LinB), 3, ATP, CoA and either AA or HAA, the production of 4 or 5, respectively, was achieved in vitro (Fig. 6B-D).The reaction was strictly dependent on the substrates (IAA, anthranilates), CoA, ATP and both enzymes.A spontaneous amidation was recognized in absence of LinB yielding negligible amounts of lindolins (1%), suggesting that the highly unstable IAA-CoA thioester quenched fast.However, the reaction was highly accelerated by LinB.In addition, an accumulation of IAA-CoA in absence of LinB or its anthranilate substrates was observed (Fig. 6C).In contrast, the intermediate IAA-CoA was scarcely detectable in the coupled enzyme reaction, indicating that LinB converts IAA-CoA as soon as it is produced by LinA.Indeed, LinA's activity is boosted twofold in the presence of LinB most likely by metabolic channeling of IAA-CoA to LinB (Fig. 6E).In sum, the ligase LinA and the transferase LinB act in concert to produce both lindolins, 4 and 5.
In contrast, LinB homologs are exclusively found in the genera of Kickxellales such as Martensiomyces spp.(e value 0; pairwise identity 61.41%), Coemansia spp.(0-5 × 10 -152 ; 49.04-63.38%),Kickxella spp.(9 × 10 -175 ; 51.15%), and Dipsacomyces spp.(8 × 10 -172 ; 56.86%) (Table 2, Additional file 40: Table S9).LinB is highly specific for this fungal order as there are no closer orthologs present in related Kickxellomycetes, other fungal orders or any eukaryotic or prokaryotic species (> 3 × 10 -18 ; < 28.07%) (Table 2).Hence, linB must have evolved in the Kickxellales ancestor cell prior to the separation from the other orders and was evolutionary conserved in all descending species.A sequence similarity network (SSN) analysis based on the LinB homologs in Kickxellales and the fairly related proteins from other kingdoms clearly demonstrates the uniqueness of the LinB enzyme class among all kingdoms of life (Fig. 8, Additional file 41: Figure S35, Additional file 42: Figure S36, Additional file 43: Figure S37).Only at a very low alignment score threshold of 26 (at which enzymes of bacteria, plants, Oomycota and Glomeromycotina collapse in a single cluster) a weak correlation between LinB proteins and predicted proteins from Glomeromycotina and plants is detectable.This might point at shikimate-O-hydroxycinnamoyl transferases of Glomeromycota or plants as a potential origin of LinB [58].Indeed, the intensive horizontal gene transfer (HGT) of the shikimate pathway between fungi, plants and prokaryotes is an important driver in eukaryotic genome evolution [59] and HGT has been reported from many EDF [60][61][62].From a natural product chemist's angle, LinB is an evolutionary unique transferase and comprises a non-canonical type of amide-bond forming enzyme in fungal secondary metabolism.

Discussion
The present work showcases the yet underestimated fungal subphylum Zoopagomycota as potent producers of bioactive natural compounds.Several species of the fungal order Kickxellales produce the IAA-derived indole amide anthranilates lindolin A and B, which possess anti-oomycete activity.The unusual amidation is NRPS-independent, but coenzyme A-mediated and relies on the activity of two enzymes, the ligase LinA and the unique transferase LinB.
Anthranilate moieties are common in bacterial benzoxazole antibiotics including nataxazole [73], carboxamycin [74], calcimycin [75] and A33853 [76] as well as in many fungal nonribosomal peptides including acetylaszonalenin, fumiquinazoline A and asperlicin [77].Benzoxazole antibiotics show diverse biological properties including anticancer and antibacterial activities [78].Anthranilate is commonly derived from l-tryptophan degradation, whilst 5-hydroxyanthanilate might be produced by an anthranilate-5-hydroxylase during anthranilate catabolism [79][80][81].Recently, a flavin-dependent monooxygenase Aha6 (UMM61384.1)was predicted to convert anthranilate into its 5-hydroxy derivative during tasikamide biosynthesis in Streptomyces tasikensis [82].However, none of the lindolin producers encodes a aha6 homolog and 5-hydroxyanthranilate might be produced E values of the most likely hits are color-coded (no hits, black; > 10 −20 , red; 10 −20 -10 −100 , yellow; < 10 −100 , green).In addition, pairwise identities of the most likely hits are color-coded (no hits, black; < 35% red, 35-40% yellow, > 40% green) Fig. 7 Phylogenetic relationship of LinA and related enzymes in other fungal orders and domains of life.For each phylum or fungal order, ten closest relatives were obtained by BLAST searching the NCBI database.The sequences were aligned using MAFFT and the tree was generated using the maximum-likelihood method of the IQ-tree webserver.All sequences are listed in Additional file 39: Table S8  by alternative shikimate anabolic or l-tryptophan catabolic pathways [83].The final step in lindolin biosynthesis is the LinA/LinBcatalyzed condensation of 3 with anthranilate.ATPdependent amide bond formation of small molecules is widely distributed in nature and is mainly catalyzed by ribosomes [84], NRPSs [85] and CoA-dependent acyl transfer systems especially during siderophore metabolism [86] and histone modification [87].In the microbial secondary metabolism, the thiotemplated amidation usually requires the tethering of the donor amino acid to a thiolated carrier protein (CP) [88].CPs  S9).Nodes are highlighted in color according to the taxonomic classification.Representative examples including LinB from Linderina pennispora and biochemically verified LinB-like transferases from plants (Avena sativa, Arabidopsis thaliana and Dianthus caryophyllus) are indicated in darker color are either embedded in an NRPS as so-called thiolation (T) domains as exemplified in the fumaryl-l-alanine biosynthesis [39] (Fig. 9A) or act as small, stand-alone CP domain proteins as shown for the nataxazole biosynthesis [73] (Fig. 9B).Kickxellomycetes follow a third, CP-independent route for thiotemplated peptide formation by using a plant-like CoA-mediated catalysis mechanism (Fig. 9C).Interestingly, isolated adenylation (A) domains of NRPSs can close peptide bonds in vitro as well [89][90][91].Similar to the native LinA/LinB-coupled system, thioester-mediated biocatalytic syntheses of dozens of different dipeptides have been demonstrated in a one-pot-reaction using two promiscuous enzymes, the A domain of the carboxylic acid reductase CAR sr coupled to the plant-derived tyramine-N-hydroxycinnamoyl acyltransferase CaAT [91].Using this approach, even post-translational modifications of proteins can be achieved.This result highlights the CoA-mediated amide formation as a powerful strategy to label or click-functionalize proteins.
The non-canonical biosynthesis of lindolins show a remarkable similarity to the structurally related 5-hydroxyanthranilate amides avenanthramides A-C (formerly known as avenalumins) from oat [48,92,93].However, the biosynthetic genes are only marginally related and may have independently evolved.Avenanthramides were initially discovered as defensive phytoalexins against the crown rust Puccinia coronata [94] but were later reidentified in oat grains [95].Avenanthramides inhibit atherosclerosis and asthma by suppression of both inflammatory cytokines and adhesion molecules in endothelial cells [96].Since 1982, their structural derivative tranilast (brand name: rizaben) has been marketed in Asia to treat allergic disorders [97] and is additionally LinA LinB a proven chemotherapeutic drug in several pre-clinical studies [98].In contrast, lindolins do not show antiproliferative effects on mammalian cell lines.However, a more profound investigation of their function during inflammation might be an objective of future works.
Tremendous effort has been conducted by comparative genomics of hundreds of EDF genomes to identify novel biosynthetic gene clusters [1,2,27].Using this promising approach, potential SM producers including the genera Mortierella [12,37], Mucor [99], Phycomyces [100], and Basidiobolus [21,22] were identified.However, previous works mainly relied on the identification of well-known SM key enzymes (e.g.NRPSs, polyketide synthases, terpene synthases and hybrids thereof ) encoded within a genetic cluster, but neglected the identification of noncanonical biosynthesis pathways or non-clustered genes.Both features apply to the lindolin biosynthesis in Kickxellales.With a few exceptions, cooperatively acting SM genes are usually co-expressed and co-located in biosynthetic gene clusters in both Ascomycota [101,102] and Basidiomycota [103][104][105].Opposingly, lindolin biosynthetic genes linA and linB are co-expressed, but not clustered.Similarly, the production of the β-carotene-derived Mucorales mating hormone trisporic acid requires several non-clustered enzymes [106][107][108] suggesting that SM biosynthetic genes might be not clustered in EDF in general-a phenomenon that is widely known in plant secondary metabolism [109].Pharmacologically important non-canonical pathways include the biosynthesis of the aforementioned anti-inflammatory avenanthramides [48], the neuroactive drug psilocybin [103], and the fly agaric toxin ibotenic acid [104], and is now expanded by the anti-oomycete lindolins from the yet underestimated fungal order Kickxellales.
Our study shows that the current strategies to identify biosynthetic routes in EDF need to be re-evaluated and the archetypical "gene cluster concept" might be reconsidered for EDF.Moreover, the non-clustered lindolin biosynthesis revealed that the metabolic potential of EDF is manifolded and much more diverse than previously predicted by genomic approaches.

Conclusion
Kickxellomycetes are a novel promising source of bioactive natural products including indole alkaloids.The noncanonical, NRPS-independent biosynthesis of the indole alkaloids lindolin A and B relies on two separate enzymes that are not encoded in a biosynthetic gene cluster.This may affect future genetic studies on secondary metabolism in EDF in general.Moreover, the metabolic potential of EDF is larger than previously recognized pointing to an underestimated reservoir for natural compounds.

Chromatographic analysis of metabolite extracts Extraction of metabolites from culture filtrate, mycelium, agar plates and soil
Culture filtrates were separated from the mycelium using Miracloth (Merck).The mycelium was rinsed with water, lyophilized to dryness, weighed to determine the fungal dry biomass and extracted with 5 mL methanol per 100 mg dry fungal biomass.The culture filtrates were adjusted to pH 6 by addition of 3 M HCl or 3 M NaOH prior to extraction with 100 mL ethyl acetate.Cultivated agar plates (25 mL) were sliced in 5 × 5 mm pieces and extracted overnight with 25 mL ethyl acetate.20 g of soil samples were extracted with 40 mL of ethyl acetate.In all extraction procedures, uninoculated liquid medium, agar or soil served as negative control.The organic phase was collected and evaporated to dryness using a rotary evaporator.The residue was dissolved in 2 mL methanol, ultrasonicated for 1 min, and centrifuged to remove contaminating particles (20,000×g, 10 min). 5 µL thereof were used for GC-MS and UHPLC measurements (methods 1 and 2, Additional file 44: Table S5).

GC-MS analysis
The compounds ILA (1), IOL (2) and IAA (3) were determined by GC-MS on a Trace 1310 gas chromatograph (Thermo Fisher Scientific) coupled with a TSQ 9000 electron impact (EI)-triple quadrupole mass spectrometer using method 1 (Additional file 44: Table S5).A 4 mm SSL GC inlet glass liner with glass wool (P/N 453A1305) and a BPX5 capillary column (30 m, 0.25 mm inner diameter, 0.25 μm film) from Trajan (SGE) was used.The column was operated with helium carrier gas (1.5 mL min −1 ) and split injection (split ratio 1:10).Total ion current (TIC) values were recorded in the mass range of 45-500 amu, with a scan time of 0.2 s. and a MS delay of 4 min.As 1 and 3 were too hydrophilic for optimal GC runs, silylation of 1-3 was conducted with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) prior to GC analysis: Methanolic fungal extracts were dried in vacuo and resuspended in 150 µL MSTFA.Samples were mixed for 15 s, ultrasonicated for 1 min and centrifuged.A volume of 1 µL of the supernatant was injected.The National Institute of Standards and Technology (NIST) Mass Spectra Search Program version 2.4 was used for comparison of the EI-MS spectra.

UHPLC-MS analysis
Compounds 3, 4 and 5 as well as IAA-CoA were analyzed on an Agilent 1290 Infinity II UHPLC instrument, coupled to a 6130 single quadrupole mass spectrometer using method 2 (Additional file 44: Table S5).Metabolite quantification was carried out using a regression curve based on the injection of gradual binary dilutions of 3, 4 and 5 from 3 to 800 µM.

Purification and structure elucidation of lindolin A (4) and B (5)
L. pennispora was grown in an upscaled culture of 12 L AMM with 1% (w/v) casamino acids and 5 mM l-tryptophan with orbital shaking at 160 rpm at 30 °C for 10 days.Metabolites were extracted from the supernatant as described above.The residue was dissolved in 130 mL dichloromethane and reduced to 40 mL with a rotary evaporator.Purification of 4 and 5 was conducted using a Büchi C-810 Flash Chromatograph and an Agilent 1200 Infinity HPLC system equipped with a diode array detector (DAD).The extract was subjected to a normal phase column (Silica gel, 40 g) on the flash chromatograph using method 3 (Additional file 44: Table S5).Four fractions (fraction I-IV) were obtained, of which fractions II and III contained 5 and 4, respectively.
Fraction III (containing 4) was evaporated to dryness and dissolved in 3 mL methanol.In four individual runs, 750 µL thereof were flash-chromatographed using a reverse phase column (C18, 12 g) running method 4 (Additional file 44: Table S5).The obtained fraction with the major peak of 4 was again dried by lyophilization and dissolved in 2 mL methanol.In several runs, aliquots of 10 µL each were subjected to semipreparative HPLC purification using method 5 (Additional file 44: Table S5).A total of 40 mg of pure 4 were obtained.Fraction II (containing 5) was evaporated to dryness and dissolved in 3.5 mL methanol.Purification was accomplished on a reverse phase column (12 g, C18) using method 6 (Additional file 44: Table S5) followed by semipreparative HPLC using method 7 (Additional file 44: Table S5).A total of 14 mg of pure 5 were obtained.MS/MS measurements were performed using a Q Exactive Plus mass spectrometer (Thermo Scientific).NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer at 300 K. DMSO-d 6 served as solvent and internal standard (δ H = 2.50 ppm and δ C = 39.52 ppm).

Heterologous protein production
linA and linB were amplified from L. pennispora cDNA using Phusion High-Fidelity DNA polymerase (NEB) and the oligonucleotides listed in Additional file 45: Table S6.The fragments were ligated into the pET28a expression vector using the restriction sites NcoI/HindIII and NheI/EcoRI (Additional file 46: Table S7), respectively.Expression was conducted in Escherichia coli SoluBL21 (linA) and BL21 (linB), cultivated in 400 mL LB medium supplemented with 50 µg mL −1 kanamycin at 37 °C until an optical density of 0.6 was reached.Then, expression was initiated by adding 1 mM IPTG.After another 16 h of incubation at 16 °C, cells were harvested by centrifugation (4000×g, 25 min) and disrupted in 5 mL lysis buffer (50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8) by ultrasonication.The enzymes were purified using the Protino Ni 2+ NTA Agarose (Macharey & Nagel) according to the manufacturer's protocol by a step gradient (10-250 mM imidazole).The proteins were subsequently concentrated and re-buffered in assay buffer (200 mM Tris, pH 7.8) using the Amicon Ultra-15 Central Filter System (30 kDa cut-off ).The protein concentration was determined by the Pierce BCA protein Assay (Thermo Fisher Scientific) using albumin as reference standard.The purity was additionally verified by SDS polyacrylamide gel electrophoresis (Additional file 33: Figure S29).The enzymes were stored at 4 °C without loss of activity for up to three weeks.

Determination of LinA activity
To determine the substrate specificity of LinA in a 200 µL scale, a modified coupled enzyme assay with myokinase, pyruvate kinase and lactate dehydrogenase according to Patel et al. was used [49,115].Final concentrations were: 200 mM Tris (pH 7.8), 20 mM MgCl 2 , 360 µM NADH, 1 mM phosphoenolpyruvate, 2.5 mM ATP, 250 µM coenzyme A, 1 U mL −1 myokinase (Merck), 1 U mL −1 pyruvate kinase (Merck), 3 U mL −1 lactate dehydrogenase (Merck) and 1 µM LinA.The reaction was initiated with 1 mM of substrates (small carboxy acids including IAA, see Fig. 5, all from Merck).Water was used as negative control.The continuous assay relies on the NADH consumption which is photometrically detected at λ = 340 nm using the ClarioStar microplate reader at 30 °C for up to 30 min.

Determination of LinB activity
To determine LinB activity, a discontinuous UHPLCbased assay was used.The final assay (500 µL) contained 200 mM Tris (pH 7.8), 20 mM MgCl 2 , 1 mM IAA-CoA and 1 µM LinB.The reaction was started with either 1 mM anthranilate (AA) or 5-hydroxyanthranilate (HAA) (Merck).Water served as negative control.After 2 h of incubation at 25 °C, the reaction was stopped by shock freezing in liquid nitrogen and lyophilization.The residue was dissolved in an equal volume acetonitrile: water (50: 50), briefly centrifuged (14,000×g, 2 min) and immediately subjected to UHPLC analysis using method 2 (Additional file 44: Table S5).A calibration curve of 4, 5 and IAA-CoA was recorded as standard for quantification.

Combined LinA/LinB assay
To produce 4 and 5 in vitro, the following assay was set up in a 500 µL scale: 200 mM Tris (pH 7.8), 20 mM MgCl 2 , 250 µM CoA, 2.5 mM ATP, 1 µM LinA, 1 µM LinB, and 1 mM AA and/or HAA.As controls, individual assay components were omitted.The reaction was initiated by addition of 1 mM IAA (3).The assay was incubated for 120 min at 25 °C.Lindolin detection was carried out by UHPLC-MS measurements as described above.

Gene cluster analysis
Comparative gene cluster analysis was performed with CAGECAT [116], an online comparative gene cluster analysis toolbox which allows homology searches between whole gene clusters or regions.The implemented clinker tool [117] was used to visualize the alignment of the genomic neighborhood of the linA and linB gene loci (± 20 kb) extracted from the genomes of eight verified lindolin-producing Kickxellales species.The minimum alignment sequence identity was set to 30%.

Phylogenetic analysis of LinA
LinA-related enzymes were identified using the BLAST search tool (matrix: BLOSUM62; gap costs: existence 11, extension 1; expect threshold: 0.05) screening the nonredundant protein sequences (nr) of NCBI for each fungal order/phylum and other domains of life separately.For each order or phylum, ten (if available) most-likely hits (according to bit score) were depicted for further analysis.Sequences with less than < 400 or > 700 amino acids were omitted as L. pennispora LinA comprises 553 aa.Moreover, biochemically verified aryl-CoA ligases were additionally included [48,53,57].Protein accession numbers, e values and pairwise identities are listed in Additional file 39: Table S8.A total of 222 amino acid sequences were aligned using MAFFT online [118] (matrix: BLOSUM62; gap open penalty: 1.53; gap extension penalty: 0.123; tree rebuilding number: 50; max iteration number: 100).The alignment was extracted to the IQ-Tree webserver [119] to generate Maximum-Likelihood trees with LG + R8 as best-fit model and a total of 1000 replicates (bootstrap support).Single branch tests were performed by SH-aLRT.A bootstrap consensus tree was computed and included 222 taxa with 763 splits.

Construction of sequence similarity networks (SSN) for LinB
LinB-related enzymes were identified using the BLAST search tool (matrix: BLOSUM62; gap costs: existence 11, extension 1; expect threshold: 0.05) screening the nonredundant protein sequences (nr) of NCBI for each fungal order/phylum and other domains of life separately.
For each order or phylum, 100 (if available) most-likely hits (according to bit score) were depicted for further analysis.Moreover, biochemically verified aryl-transferases were additionally included [48,58,120].Protein accession numbers, e values and pairwise identities are listed in Additional file 40: Table S9.SSNs were constructed using the Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST) [121].Two different SSNs were generated with EFI-EST using the default setting of a −log(e value) = 5 for all-by-all BLAST to calculate similarities and edge alignment score similarities resulting in 505 nodes and > 25,000 edges.The sequences in the generated SSN were restricted to a sequence length of 400-600 amino acids (Additional file 41: Figure S35, Additional file 42: Figure S36, Additional file 43: Figure S37).The alignment score threshold was set to 60 (corresponding to 35% sequence identity) or 26 (25%) for figure panels 8A and 8B, respectively.Data was visualized in Cytoscape v. 3.10.2[122] using a "prefuse force directed layout" with the option "all nodes: alignment_score".

Fig. 2
Fig. 2 Production of lindolin A and B in Kickxellales.Pictures of agar plates and extracted ion chromatograms (EIC) of metabolite crude extracts of Linderina pennispora, Martensiomyces pterosporus and Coemansia furcata grown in liquid meat medium (meat, A) or potato dextrose broth (PDB, B) at 160 rpm for 6 d.EICs were recorded at m/z 293 [M−H] − and m/z 309 [M−H] − for 4 (red trace) and 5 (blue trace), respectively.Non-inoculated medium served as negative controls.Arrows indicate traces of 4 or 5

Fig. 3
Fig.3Secretion of lindolins under natural conditions.Linderina pennispora, Martensiomyces pterosporus and Coemansia furcata were submersely grown in liquid meat medium under agitation.Crude extracts of culture supernatants (A) and mycelia (B) were chromatographed.In addition, cultures were emersely cultivated on meat agar plates (C) and in potting soil supplemented with d-glucose (D).Authentic standards of 4 and 5 served as controls (E).Pictures of the soil cultures are shown above the chromatograms (Aerial mycelium is mainly visible for L. pennispora and C. furcata.Minor white spots in the control plates are part of the soil substrate).Chromatograms were recorded by UHPLC-MS and overlaid extracted ion chromatograms were shown for m/z 293 [M−H] − and m/z 309 [M−H] − for 4 (red trace) and 5 (blue trace), respectively.Non-inoculated media or soil served as negative controls.Asterisks indicate an unrelated compound that is also present in the soil negative control.Note, that lindolin A (4) is absent in the mycelium, but is the predominant secreted compound in the supernatant, agar plates and soil

Fig. 6
Fig. 6 Biocatalytic lindolin production in vitro.A Schematic representation of the two-step biosynthesis to lindolins.The reaction was initiated by addition of the substrate anthranilic acid (AA) (B and C) or 5-hydroxyanthranilic acid (HAA) (D) and incubated at 25 °C for 2 h.The amount of 4 (B), IAA-CoA (C) and 5 (D) was chromatographically quantified.Representative EICs are given for 4, IAA-CoA and 5 at m/z 293 [M−H] − , m/z 923 [M−H] − , and m/z 309 [M−H]− .Note, that the CoA ester leads to slightly broadened peaks as described elsewhere[51].E Enzyme-coupled determination of LinA activity in presence and absence of LinB

Fig. 8
Fig. 8 Sequence similarity networks (SSN) for LinB.A LinB homologs are depicted in a protein sequence similarity network with an alignment score threshold of 60 (correlating to 35% sequence identity) resulting in a network with distinctly separated clusters corresponding to the different phyla.B LinB homologs are depicted in a protein sequence similarity network with an alignment score threshold of 26 (correlating to 25% sequence identity) and showing the sole, but weak correlation between the nodes of LinB sequences from Kickxellales to nodes of other phyla, especially to Glomeromycotina and plants.Each node represents a member of up to 100 closest LinB orthologs in each phylum or fungal order according to the BLAST search (see Additional file 40: Table S9).Nodes are highlighted in color according to the taxonomic classification.Representative examples including LinB from Linderina pennispora and biochemically verified LinB-like transferases from plants (Avena sativa, Arabidopsis thaliana and Dianthus caryophyllus) are indicated in darker color

Fig. 9
Fig. 9 Thiotemplated amidation reactions in microbes.The thiotemplated amidation in secondary metabolism requires an active thioester prior to peptide transfer.The donor acid is usually bound to a carrier protein (CP) that is either part of an NRPS (A) or a freestanding CP domain protein (B).In contrast, lindolin biosynthesis in Kickxellomycetes rely on a CP-independent but CoA-mediated amide formation (C).A adenylation domain, ACP acyl carrier protein, C condensation domain, NatC1/2 freestanding CPs, NatL1/2 aryl-AMP synthetases, NatS aryl-ACP synthases, T thiolation domain (NRPS-bound CP).Donor and acceptor acids are highlighted in blue and red, respectively

Table 1
Heterologously expressed genes from Linderina pennispora and biochemical function of the corresponding enzymes

Table 2
Blast analyses of the closest relatives of the ligase LinA and the transferase LinB