Natural products derived from micro-organisms represent an incomparable source of chemical novelty, and the structural and chemical diversity which can be obtained is greater when genetically diverse strains are considered. Our collection of 45.000 actinomycetes is enriched in uncommon genera, and in this work we focused on the genus Actinospica, which we described in 20061. Our early finding of NRPS and PKS genes in Actinospica strains2 and the presence in the genome of A. robiniae (NZ_KI632511.1) of >20 genetic clusters related to secondary metabolism suggest a potential to produce bioactive metabolites. We therefore investigated the diversity and metabolic potential of 22 isolates derived from our strain library.

This work received support from the European Commission under grant agreement 664588 (NOMORFILM project). B.D.O was supported by a training grant from Regione Lazio (Torno Subito program).

1. Cavaletti et al. (2006) Int J Syst Evol Microbiol 56, 1747-1753; 2. Busti et al.(2006) Microbiology 152, 675-683; 3. Wang et al. (2016) Nature Biotechnol 34, 828-837; 4. Wu et al. (2011) Antimicrobial Ag Chemother55, 974-982; 5. Yaginuma et al. (1987) J Antibiotics 40, 239-241.

Molecular networking evidenced unique metabolites, which were confirmed to be structurally novel. With just a few analyzed strains, the genus Actinospicabears promise for novel metabolites.

Agar-diffusion tests showed that extracts from four strains (G8, G23, G25 and G26) were activevs S.aureus. The active extracts from strains G8 and G23 were also highlighted by GNPS analysis as harboring unique metabolites. Thus, the bioactive metabolites were purified and characterized.

G23 (EtOH) G20, A.acidiphila (EtOH+SPE)

G25, G26 (SPE)

G8 (EtOH+SPE)

Metabolites from strain G23

Activity in the mycelium extract was due to the new Calcimycin-like metabolite 1 as the major component. Despite the numerous representatives of this class, metabolite 1 from Actinospica is the first one possessing a methyl group at position 9.

4

5

RT: 5.51 - 9.46 SM: 3B

6.0 6.5 7.0 7.5 8.0 8.5 9.0

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

8.51538.04

8.28523.99

6.54540.276.24

526.29

7.73541.88

6.90526.20 7.98

538.107.27

526.185.77

544.758.90

540.19

NL: 1.18E4

Base Peak m/z= 510.00-545.00 F: ITMS + c ESI Full ms [300.00-2000.00] MS SNT_48hRT

Furthercongeners

G8_1G8_2

G8_1G8_2

G8_3G8_4

G8_3G8_4

1/2

3/4

Metabolites from strain G8The bioactivity was associated to a complex of up to 16 related metabolites. Structure elucidation of the most stable and abundant congeners revealed new ansamycin-like metabolites characterized by a linear or branched lipophilic chain, longer than that observed so far in this class. Compounds 1-2appear more active than 3-4.

MS/MS based clustering coupled with 16S data can single out strain-specific metabolites (i.e., derivingfrom a single strain or strains with identical 16S sequence), absent in a reference database of actinomycete-derived metabolite fingerprints.

Investigating the metabolic potential of the rare actinomycete genus ActinospicaPAOLO MONCIARDINI*°, SONIA I. MAFFIOLI*°, ARIANNA TOCCHETTI°, ALICE BERNASCONI°, CRISTINA BRUNATI*, BARBARA D’ORIO°, SILVIA PESSINA°, MARGHERITA SOSIO*°, STEFANO DONADIO*°*KtedoGen srl, Milano, Italia; °Naicons srl, Milano, Italia

Introduction

1. Strains grownon plates

2. Flask cultures

4b. Bioassay5. Metabolite clusterization

based on MS/MS data3

3. EtOH (mycelium) or SPE (clearedbroth) extracts

4a. LC/MS analysis

Workflow

Molecular Networking Bioactivity

Neighbor-Joining tree based on 16S phylogeny showing the placement of the investigated strains (green) in comparisonwith described species (yellow) and with GenBanksequence entries deriving from undescribed isolates. Bootstrap values (1000 replicates) >70% are shown atnodes. Scale bar: 1 inferred substitution per 100 nt.

Based on 16S phylogeny, the isolates representdifferent lines of descent within Actinospica, including some distantly related to describedspecies.

0.01

Actinospica sp.G4

Actinospica durhamensis KJ445732.1

Isolate Aac35 AB180777

Actinospica spp.G1-G5-G7-G11

Actinospica sp.G12

Isolate Aac-2 AB180765

Actinospica spp. G17-G18

Gamma25 AJ865864

Actinospica robiniae AJ865863

Actinospica spp.G3-G6-G9-G10-G13-G14-G15-G16-G19

Isolate Aac33 AB180776

Actinospica sp.G23

Actinospica sp.G25-G26

Isolate Aac50 AB180784

Isolate Aac29 AB180771

Isolate Aac61 AB180788

Isolate Aac99 AB180796

Isolate Aac49 AB180782

Isolate Aac57 AB180786

Gamma4 AJ865862

Actinospica sp.G20

Actinospica acidiphila AJ865861

Isolate Aac-3 AB180772

Isolate Aac85 AB180794

Actinospica sp.G8

Isolate Aac-15 AB180762

Isolate Aac25 AB180769

1000

906756

873

853

742

909

964

Strain Diversity

Acknowledgements

Conclusions

References