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5.1 Abstract

Six out of 57 bioactive filamentous actinobacteria, notably isolates H9 and H45, isolated from high altitude Atacama Desert soils were found to produce new specialized metabolites. Large scale fermentation on two production media followed by chromatographic purification and LCMS and NMR analyses led to the isolation of six new and one known diene from isolate H45. The structures of the new compounds, named lentzeosides A-F, were confirmed by HRESIMS and NMR analyses. The purified lentzeosides were found to inhibit HIV-1 integrase activity. Isolate H9 produced a prospective novel peptide and polyunsaturated components based on LCMS and UV spectrophotometric traces. Polyphasic studies designed to establish the taxonomic status of isolates H45 and H9 showed that they had chemotaxonomic and morphological features consistent with their classification in the genera Lentzea and Streptomyces, respectively. The Lentzea strain was found to be most closely related to Lentzea kentuckyensis NRRL B-24416^T based on 16S rRNA gene sequence data but was distinguished readily from the latter by a low level of DNA:DNA relatedness and by a broad range of phenotypic properties. Analysis of 16S rRNA gene sequences showed that isolate H9 formed a distinct phyletic line in the Streptomyces gene tree. Multilocus sequence analysis based on five housekeeping gene alleles underpinned the separation of the strain from all of its nearest phylogenetic neighbours as did corresponding phenotypic data. In light of these results it is proposed that isolates H45 and H9 be recognized as new species of Lentzea and Streptomyces, respectively, namely as Lentzea chajnantorensis sp.

nov. and Streptomyces aridus sp. nov., with the type strains H45^T (NCIMB 14966^T

=NRRL B-65282^T) and H9^T (NCIMB 14965^T =NRRL B-65268^T), respectively.

5.2 Introduction

Novel filamentous actinobacteria tend to feature in bioprospecting campaigns due to their ability to synthesize new natural products that belong to novel structural classes and have new modes of action (Goodfellow and Fiedler 2010; Barka et al. 2016). The premise that novel actinobacteria from extreme biomes are a rich source of new bioactive compounds

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has gained credence with the discovery that isolates from arid and extreme hyper-arid Atacama Desert soils synthesize new chemical structures with potent biological activity (Bull and Asenjo 2013; Bull et al. 2016), as exemplified by Streptomyces leeuwenhoekii strains (Busarakam et al. 2014; Rateb et al. 2011b) which synthesize the atacamycins (Nachtigall et al. 2011), chaxalactins (Rateb et al. 2011a) and chaxapeptin, a new lasso peptide (Elsayed et al. 2015). The type strain of this species has the makings of a key model organism in the search for novel natural products as its whole-genome sequence contains 38 biosynthetic gene clusters that encode for known and undiscovered specialized metabolites (Castro et al. 2015; Gomez-Escribano et al. 2015).

The present study was designed to build upon and extend the pioneering bioprospecting studies on Atacama Desert actinobacteria as outlined above. To this end, extracts of 57 actinobacterial strains isolated from high altitude Atacama Desert soils and 4 from corresponding extreme hyper-arid soils that were known to inhibit the growth of Bacillus subtilis and/or Escherichia coli strains were the subject of chemical analyses which showed that six of them produced new compounds, notably those synthesized by isolates H9 and H45; these strains were the subject of polyphasic studies which showed that they formed the nuclei of novel species of Streptomyces and Lentzea, respectively.

Detailed chemical screening of extracts from the novel Lentzea strain led to the isolation of six novel and one known diene, as well as monoene glycosides. The novel dienes were found to inhibit HIV-1 integrase activity.

5.3 Materials and Methods

5.3.1 Selection of isolates for bioactivity screening

Sixty one strains found to inhibit the growth of Bacillus subtilis and/or Escherichia coli wild type strains in primary plug assays (see Chapter 3) were selected for bioactive compound screening in the Marine Biodiscovery Centre at the University of Aberdeen.

The source of the strains, their assignment to colour-groups and their ability to inhibit the growth of a panel of wild type and Bacillus subtilis reporter strains are shown in Table 5.1, details of the Chajnantor and Lomas Bayas sampling sites can be found in Table 2.1 in Chapter 2.

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Table 5.1 Source, colour-group assignment and bioactivity profiles of Atacama Desert isolates selected for bioactive compound screening.

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*Colour-group: 1-80, Number of colour-groups; ^M, multi-membered colour-group; ^(1-31), number of isolates in multi-membered colour groups; ^S, single-membered colour-groups, see Appendix 1 for details.

*Panel of wild type strains included in standard plug assays: a, Escherichia coli; b, Pseudomonas fluorescens; c, Bacillus subtilis; d, Staphylococcus aureus; e, Saccharomyces cerevisiae. See Chapter 2 for details.

* Bacillus subtilis reporter strains: Phi105^CH (inhibition of DNA synthesis); ypuA^ER (inhibition of cell envelope synthesis); yvqI^ER (inhibition of cell wall synthesis); yvgS^ER (inhibition of RNA synthesis); yheH^ER (inhibition of protein synthesis) and yjaX^ER (inhibition of fatty acid synthesis). (see Chapter 2 for details).

5.3.2 Preliminary detection of bioactive compounds

Fermentation conditions. The 61 strains were grown in shake flasks at 180 revolutions per minute (rpm) for 10 days at 28 ^oC in yeast extract-malt extract (Shirling and Gottlieb 1966), media 19 and 410 (Goodfellow and Fiedler 2010) and starch-casein broths (Küster and Williams 1964) which contained a gram of Amberlite beads (Sigma-Aldrich, Gillingham Dorset, UK) per 50 ml of broth for specialized metabolite adsorption.

Extraction of metabolites. The Amberlite beads were separated from the production broths by centrifugation at 4000 rpm for 5 minutes, washed 4-5 times with sterile distilled water, soaked in 50 ml of methanol and sent to Professor Marcel Jaspars at Aberdeen University for preliminary chemical analyses. In Aberdeen, samples were extracted twice with methanol and acetone (2:1, v/v) and the corresponding extracts combined and concentrated in vacuo. The resultant preparations were purified by using the Biotage SP1 flash system (Biotage, USA) with a reversed phase 40iM cartridge (KP-C18-HSTM, 40 x 150 mm) with a gradient of methanol in water (5-100% in 12 column volumes), detection of compounds was carried out at 220 and 254 nm.

Chemical analyses of extracts. Each extract was dissolved in methanol to give a final concentration of 0.5mg/mL, filtered and submitted for liquid chromatographic/mass spectrometric analysis (LCMS). High resolution mass spectral data were obtained using a Thermo Instruments MS system (LTQ XL/ LTQ Orbitrap Discovery, ThermoFiscer, Germany) coupled to a Thermo Instruments HPLC system (Accela PDA detector, Accela